Use of jagged 1/frizzled 4 as a cell surface marker for isolating human cardiac ventricular progenitor cells

ABSTRACT

The present invention provides Jagged 1 and Frizzled 4 as cell surface markers for isolating human cardiomyogenic ventricular progenitor cells, in particular progenitor cells that preferentially differentiate into cardiac ventricular muscle cells. Thus, the invention provides human ventricular progenitor (HVP) cells. The invention provides in vitro methods of the separation of Islet 1+ Jagged 1+ ventricular progenitor cells and/or Islet 1+/Frizzled 4+ ventricular progenitor cells and/or Islet 1+/Jagged 1+/Frizzled 4+ ventricular progenitor cells, and the large scale expansion and propagation thereof. Large clonal populations of isolated Jagged 1+ and/or Frizzled 4+ventricular progenitor cells are also provided. Methods of in vivo use of Jagged 1+ and/or Frizzled 4+ ventricular progenitor cells for cardiac repair or to improve cardiac function are also provided. Methods of using the Jagged 1+ and/or Frizzled 4+ ventricular progenitor cells for cardiac toxicity screening of test compounds are also provided.

RELATED APPLICATIONS

This application claims the benefit of the priority date of U.S.Provisional Application No. 62/040,892, which was filed on Aug. 22,2014, and U.S. Provisional Application No. 62/194,016, which was filedon Jul. 17, 2015. The content of these provisional applications ishereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Heart failure, predominantly caused by myocardial infarction, is theleading cause of death in both adults and children worldwide and isincreasing exponentially worldwide (Bui, A. L. et al. (2011) Nat. Rev.Cardiol. 8:30-41). The disease is primarily driven by the loss ofventricular muscle that occurs during myocardial injury (Lin, Z. and Pu,W. T. (2014) Sci. Transl. Med. 6:239rv1) and is compounded by thenegligible ability of the adult heart to mount a regenerative response(Bergmann, O. et al. (2009) Science 324:98-102; Senyo, S. E. et al.(2013) Nature 493:433-436). Although heart transplantation can becurative, the markedly limited availability of human heart organ donorshas led to a widespread unmet clinical need for a renewable source ofpure, mature and functional human ventricular muscle tissue (Segers, V.F. M. and Lee, R. J. (2008) Nature 451:937-942; Später, D. et al. (2014)Development 141:4418-4431).

Human pluripotent stem cells (hPSCs) offer the potential to generatelarge numbers of functional cardiomyocytes for potential clinicalrestoration of function in damaged or diseased hearts. Transplantationof stem cells into the heart to improve cardiac function and/or toenrich and regenerate damaged myocardium has been proposed (see e.g.,U.S. Patent Publication 20040180043). Combination therapy, in whichadult stem cells are administered in combination with treatment withgrowth factor proteins has also been proposed (see e.g., U.S. PatentPublication 20050214260).

While cell transplantation into the heart offers a promising approachfor improving cardiac function and regenerating heart tissue, thequestion of what type(s) of cells to transplant has been the subject ofmuch investigation. Types of cells investigated for use in regeneratingcardiac tissue include bone marrow cells (see e.g., Orlic, D. et al.(2001) Nature 410:701-705; Stamm, C. et al. (2003) Lancet 361:45-46;Perin, E. C. et al. (2003) Circulation 107:2294-2302), adult stem cells(see e.g., Jackson, K. A. et al. (2001) J. Clin. Invest. 107:1395-1402),bone marrow-derived mesenchymal stem cells (see e.g., Barbash, I. M. etal. (2003) Circulation 108:863; Pettinger, M. F. and Martin, B. J.(2003) Circ. Res. 95:9-20), bone marrow stromal cells (Bittira, B. etal. (2003) Eur. J. Cardiothorac. Surg. 24:393-398), a combination ofmesenchymal stem cells and fetal cardiomyocytes (see e.g., Min, J. Y. etal. (2002) Ann. Thorac. Surg. 74:1568-1575) and a combination of bonemarrow-derived mononuclear cells and bone marrow-derived mesenchymalstem cells (see e.g., U.S. Patent Publication 20080038229).Dedifferentiation of adult mammalian cardiomyocytes in vitro to generatecardiac stem cells for transplantation has also been proposed (see e.g.,U.S. Patent Publication 20100093089).

A significant advancement in the approach of cell transplantation toimprove cardiac function and regenerate heart tissue was theidentification and isolation of a family of multipotent cardiacprogenitor cells that are capable of giving rise to cardiac myocytes,cardiac smooth muscle and cardiac endothelial cells (Cai, C. L. et al.(2003) Dev. Cell. 5:877-889; Moretti, A. et al. (2006) Cell127:1151-1165; Bu, L. et al. (2009) Nature 460:113-117; U.S. PatentPublication 20060246446). These cardiac progenitor cells arecharacterized by the expression of the LIM homeodomain transcriptionfactor Islet 1 (Isl1) and thus are referred to as Isl1+ cardiacprogenitor cells. (Ibid). In contrast, Isl1 is not expressed indifferentiated cardiac cells. Additional markers of the Isl1+ cardiacprogenitor cells that arise later in differentiation than Isl1 have beendescribed and include Nkx2.5 and flk1(see e.g., U.S. Patent Publication20100166714).

The renewal and differentiation of Isl1+ cardiac progenitor cells hasbeen shown to be regulated by a Wnt/beta-catenin signaling pathway (seee.g., Qyang, Y. et al. (2007) Cell Stem Cell. 1:165-179; Kwon, C. et al.(2007) Proc. Natl. Acad. Sci. USA 104:10894-10899). This has led to thedevelopment of methods to induce a pluripotent stem cell to enter theIsl1+ lineage and for expansion of the Isl1+ population throughmodulation of Wnt signaling (see e.g., Lian, X. et al. (2012) Proc.Natl. Acad. Sci. USA 109:E1848-57; Lian, X. et al. (2013) Nat. Protoc.8:162-175; U.S. Patent Publication 20110033430; U.S. Patent Publication20130189785).

While human pluripotent stem cells hold great promise, a significantchallenge has been the ability to move from simply differentiation ofdiverse cardiac cells to forming a larger scale pure 3D ventricularmuscle tissue in vivo, which ultimately requires vascularization,assembly and alignment of an extracellular matrix, and maturation.Toward that end, a diverse population of cardiac cells (atrial,ventricular, pacemaker) has been coupled with artificial anddecellurized matrices (Masumoto, H. et al. (2014) Sci. Rep. 4:5716; Ott,H. C. et al. (2008) Nat. Med. 14:213-221; Schaaf, S. et al. (2011) PLoSOne 6:e26397), vascular cells and conduits (Tulloch, N. L. et al. (2011)Circ. Res. 109:47-59) and cocktails of microRNAs (Gama-Garvalho, M. etal. (2014) Cells 3:996-1026) have been studies, but the goal remainselusive.

While the identification of Isl1 as a marker of cardiac progenitor cellswas a significant advance, since Isl1 is an intracellular protein it isnot a suitable marker for use in isolating large quantities of viablecells. Rather, a cell surface marker(s) is still needed. Furthermore,Isl1 as a marker identifies a population that can differentiate intomultiple cell types within the cardiac lineage, and thus there is stilla need for markers that identify cardiac progenitor cells that arebiased toward a particular cell type within the cardiac lineage, inparticular for progenitor cells that differentiate into ventricularcells. Accordingly, there is still a great need in the art foradditional markers of cardiac progenitor cells, in particularcell-surface markers of cardiac progenitor cells, that predominantlygive rise to cardiomyocytes and that would allow for rapid isolation andlarge scale expansion of cardiomyogenic progenitor cells. Furthermore,there is still a great need in the art for methods and compositions forisolating cardiac ventricular progenitors, which differentiate intoventricular muscle cells in vivo, thereby allowing for transplantationof ventricular progenitors or ventricular muscle cells in vivo toenhance cardiac function.

SUMMARY OF THE INVENTION

This invention describes the use of Jagged 1 (JAG1) or Frizzled 4 (FZD4)as a cell surface marker for isolating human cardiac progenitor cells.Furthermore, these human cardiac progenitor cells are biased toward theventricular lineage such that they differentiate predominantly intoventricular muscle cells both in vitro and in vivo. That is, thesecardiac progenitor cells can be cultured under conditions in vitro suchthat they are biased toward the ventricular lineage and thus are humanventricular progenitor (HVP) cells. Moreover, when introduced into theventricular region of the heart in a subject, these progenitor cellsdifferentiate almost exclusively into ventricular muscle cells thatfunction according to their ventricular programming. In particular, thehuman ventricular progenitor cells provided herein utilize a cellautonomous pathway by which these cells can build a pure 3-Dvascularized, functional and mature ventricular cell wall in vivo on thesurface of normal murine kidney or heart, thereby allowing fororgan-on-organ in vivo tissue engineering.

Using Islet 1 (ISL1) as a marker, a scalable two-step culture protocolfor generating HVPs has been developed and cell surface markers(JAG1/FZD4) have been identified that allow the generation andpurification of billions of pure HVPs from human pluripotent stem cells(hPSCs). These HVPs can also be identified in the 4 week human fetalheart ventricular chambers.

Transplantation of the ventricular progenitor cells provided hereinproduces a pure, functional and mature human ventricular muscle organ oflarge size (e.g., twice the size of the murine heart) that can generateforce, respond to catecholamines, lose automaticity, contain T tubulesand display hypertrophic growth of adult rod-shaped cells by 5 monthspost-transplantation. Thus, human ventriculogenesis can be achieved viaa cell autonomous pathway driven by the purified ISL1/JAG1/FZD4 humanventricular progenitors provided herein. These HPVs provided hereinallow for new in vivo models of human cardiac disease in murine-humanchimeras and for the development of organ-on-organ regenerativetherapeutic strategies for cardiac disease.

This identification of a key cell surface marker of cardiac ventricularprogenitor cells allows for easy and rapid isolation of the cells.Furthermore, determination of culture conditions for expansion andventricular lineage bias of the cells allows for the preparation oflarge cultures (a billion or more cells) of a clonal population ofcardiac ventricular progenitor cells. These cells can be used, forexample, to improve function in a damaged heart in a subject,particularly damage in the ventricular region. The progenitor cells canbe transplanted in vivo for differentiation into ventricular cells insitu or, alternatively, a heart muscle patch, comprising ventricularmuscle cells, can be prepared in vitro from the progenitors forsubsequent transplantation in vivo. The cells also can be used, forexample, in in vitro toxicity screening assays to evaluate the cardiactoxicity of test compounds, as well as for biochemical studies toidentify relevant pathways used in cardiac maturation anddifferentiation.

Thus, the invention provides for the first time human cardiacventricular progenitor cells in purified form. The human cardiacventricular progenitors are capable of differentiation into ventricularmuscle cells in vitro and in vivo. These progenitor cells can beexpanded to large numbers of cells in vitro and when transplanted intothe ventricular region of the heart in vivo they differentiateessentially exclusively into ventricular muscle cells. Still further,the cells have the capacity to migrate in vivo to different sites and,when transplanted in vivo the cells does what they are programmed to doas a ventricular cell (as opposed to a cardiac myocyte which simplycontracts). Thus, the ventricular progenitor cells can be grafted tonative tissue to enhance ventricular function and have the ability tocall in vasculature into the new ventricular tissue. Using the RNA-seqtechnique combined with a robust cardiac differentiation protocol,transcriptional expression at a genome-scale level at different timepoints of hPSC differentiation was performed. These experiments led tothe identification of Jagged 1 and Frizzled 4 as cell surface markersfor Isl1+ cardiomyogenic progenitor cells derived from hPSCs.Co-expression of Isl1 and Jag 1, or Isl1 and Frzd 4, on cardiomyogenicprogenitor cells in vitro was demonstrated. Furthermore, it has beendemonstrated that Jag1 and Is11 are co-expressed in the week 4 humanfetal heart in vivo. Still further, after transplantation of purifiedhuman Isl1+ Jag1+ cardiomyogenic progenitor cells into normal or injuredhearts in mice, enriched human Isl1+ Jag1+ cells gave rise to cTnT+cardiomyocytes demonstrating the cardiomyogenic nature of the Isl1+Jag1+ progenitor cells. In these in vivo transplantation studies, largergrafts were observed in the injured hearts transplanted with Isl1+ Jag1+cardiomyogenic progenitor cells, as compared to normal hearts,demonstrating the capacity of the Isl1+ Jag1+ cardiomyogenic progenitorcells for cardiomyocyte regeneration.

Still further, the RNA-seq experiments identified additional potentialsurface markers, including the following markers for mesoderm cellsexpressing brachyury: FZD10, CD48, CDID, CD8B, IL15RA, TNFRSF1B,TNFSF13, ICOSLG, SEMA7A, SLC3A2, SDC1 and HLA-A; and the followingmarkers for cardiac mesoderm mesp1 positive cells: CXCR4, ANPEP, ITGAS,TNFRSF9, FZD2, CDID, CD177, ACVRL1, ICAM1, LICAM, NGFR, ABCG2, FZD7,TNFRSF13C and TNFRSF1B; and the following markers for cardiac progenitorcells: PDGFRA, LIFR (CD118), TNFSF9 and FGFR3. Any of these additionalcardiac progenitor markers can be used in the methods of the inventionto isolate progenitors at different stages of differentiation. Inparticular, the cardiac progenitor markers PDGFRA, LIFR (CD118), TNFSF9or FGFR3 can be used in a similar manner to JAG1 and FZD4, as describedherein, for isolation of cardiomyogenic progenitors.

Accordingly, in one aspect, the invention pertains to a method forisolating human cardiac ventricular progenitor cells, the methodcomprising:

-   -   contacting a culture of cells containing human cardiac        progenitor cells with one or more agents reactive with Jagged 1        and/or Frizzled 4; and separating Jagged 1 and/or Frizzled 4        reactive positive cells from non-reactive cells to thereby        isolate human cardiac ventricular progenitor cells.        In one embodiment, the human cardiac progenitor cells are        contacted both with an agent reactive with Jagged 1 and with an        agent reactive with Frizzled 4 to thereby separate Jagged 1 and        Frizzled 4 reactive positive cells from non-reactive cells.

Preferably, the human cardiac progenitor cells are Islet 1+ humancardiac progenitor cells. In another embodiment, the culture of cells isalso contacted with an agent reactive with Islet 1; and Jagged 1reactive/Islet 1 reactive positive cells, or Frizzled 4 reactive/Islet 1reactive cells, are separated from non-reactive cells to thereby isolatecardiac ventricular progenitor cells. The culture of cells can besimultaneously contacted with the agent reactive with Jagged 1 and/orFrizzled 4 and the agent reactive with Islet 1. Alternatively, theculture of cells can be contacted with the agent reactive with Islet 1before contacting with the agent reactive with Jagged 1 and/or Frizzled4. Alternatively, the culture of cells can be contacted with the agentreactive with Jagged 1 and/or Frizzled 4 before contacting with theagent reactive with Islet 1. In another embodiment, the human cardiacventricular progenitor cells are further cultured and differentiatedsuch that they express the ventricular marker MLC2v.

In another aspect, the invention pertains to a method for isolatinghuman cardiac ventricular progenitor cells, the method comprising:

-   -   culturing human pluripotent stem cells under conditions that        generate cardiac progenitor cells to obtain a cultured cell        population;    -   contacting the cultured cell population with one or more agents        reactive with Jagged 1 and/or Frizzled 4; and    -   separating Jagged 1 and/or Frizzled 4 reactive positive cells        from non-reactive cells to thereby isolate human cardiac        ventricular progenitor cells.

In one embodiment, the human cardiac progenitor cells are contacted bothwith an agent reactive with Jagged 1 and with an agent reactive withFrizzled 4 to thereby separate Jagged 1 and Frizzled 4 reactive positivecells from non-reactive cells.

In one embodiment, the culture of cells is also contacted with an agentreactive with Islet 1; and Jagged 1 reactive/Islet 1 reactive positivecells, or Frizzled 4 reactive/Islet 1 reactive positive cells, areseparated from non-reactive cells to thereby isolate human cardiacventricular progenitor cells. The culture of cells can be simultaneouslycontacted with the agent(s) reactive with Jagged 1 and/or Frizzled 4 andthe agent reactive with Islet 1. Alternatively, the culture of cells canbe contacted with the agent reactive with Islet 1 before contacting withthe agent(s) reactive with Jagged 1 and/or Frizzled 4. Alternatively,the culture of cells can be contacted with the agent(s) reactive withJagged 1 and/or Frizzled 4 before contacting with the agent reactivewith Islet 1. In another embodiment, the human cardiac ventricularprogenitor cells are further cultured and differentiated such that theyexpress the ventricular marker MLC2v.

In the methods for isolating human cardiac ventricular progenitor cells,various types of agents that bind to Jagged 1 or Frizzled 4 can be usedas the agent(s) reactive with Jagged 1 or Frizzled 4. For example, inone embodiment, the agent reactive with Jagged 1 or Frizzled 4 is ananti-Jagged 1 antibody or an anti-Frizzled 4 antibody, such as amonoclonal antibody. In another embodiment, the agent reactive withJagged 1 or Frizzled 4 is a soluble Jagged 1 ligand or Frizzled 4ligand, such as a Jagged 1 ligand fusion protein or a Frizzled 4 ligandfusion protein. For example, the agent reactive with Jagged 1 cancomprise the Jagged 1 ligand Notch-1, such as a soluble Notch-1 fusionprotein (e.g., a Notch-1/Ig fusion protein).

In the methods for isolating human cardiac ventricular progenitor cells,various types of separation methods can be used to separate Jagged 1and/or Frizzled 4 reactive positive cells (or Jagged 1/Islet 1 orFrizzled 4/Islet 1 reactive positive cells) from non-reactive cells. Forexample, in one embodiment, the reactive positive cells are separatedfrom the non-reactive cells by fluorescence activated cell sorting(FACS). In another embodiment, the reactive positive cells are separatedfrom the non-reactive cells by magnetic activated cells sorting (MACS).

In yet another aspect, the invention pertains to a method of obtaining aclonal population of human cardiac ventricular progenitor cells, themethod comprising:

-   -   isolating a single Jagged1+ and/or Frizzled 4+ human cardiac        ventricular progenitor cell; and    -   culturing the single Jagged1+ and/or Frizzled 4+ human cardiac        ventricular progenitor cell under conditions such that the cell        is expanded to at least 1×10⁹ cells to thereby obtain a clonal        population of human cardiac ventricular progenitor cells.

In one embodiment, the single Jagged1+ and/or Frizzled 4+ human cardiacventricular progenitor cell is Islet 1 positive, Nkx2.5 negative andflk1 negative at the time of initial culture. The single Jagged1+orFrizzled 4+ human cardiac ventricular progenitor cell can be isolated bymethods such as those described above (e.g., FACS or MACS). The singleJagged1+ and/or Frizzled 4+ human cardiac ventricular progenitor cellcan be isolated using a reagent(s) reactive with Jagged 1 and/orFrizzled 4, such as those described above (e.g., anti-Jagged 1antibodies, anti-Frizzled 4 antibodies, soluble Jagged 1 ligands orsoluble Frizzled 4 ligands, such as ligand fusion proteins). Uponfurther culture and differentiation, the clonal population of humancardiac ventricular progenitor cells can express the ventricular markerMLCV2.

In a preferred embodiment, the single Jagged 1+ and/or Frizzled 4+ humancardiac ventricular progenitor cell is cultured in vitro underconditions such that the cell is biased toward ventriculardifferentiation. For example, the single Jagged1+ and/or Frizzled 4+human cardiac ventricular progenitor cell can be cultured in CardiacProgenitor Culture (CPC) medium (80% advanced DMEM/F12 supplemented with20% KnockOut Serum Replacement, 2.5 mM GlutaMax and 100 μg/ml VitaminC), which allows for differentiation of the cells into ventricular cellsexpressing the MLC2v ventricular marker. In various embodiments, thesingle Jagged 1+ and/or Frizzled 4+ human cardiac ventricular progenitorcell is expanded to a clonal population of, for example, at least 1×10⁹cells, at least 2×10⁹ cells, at least 5×10⁹ cells or at least 10×10⁹cells.

Accordingly in another aspect, the invention pertains to a clonalpopulation of isolated Jagged 1+ and/or Frizzled 4+ human cardiacventricular progenitor cells. In various embodiments, this clonalpopulation comprises, for example, at least 1×10⁹ cells, at least 2×10⁹cells, at least 5×10⁹ cells or at least 10×10⁹ cells. In a preferredembodiment, this clonal population comprises at least 1×10⁹ Jagged 1+human cardiac ventricular progenitor cells or at least 1×10⁹ Frizzled 4+human cardiac ventricular progenitor cells.

In yet another aspect, the invention pertains to a method of enhancingcardiac function in a subject using the Jagged 1+ and/or Frizzled 4+human cardiac ventricular progenitor cells described herein. Forexample, in one embodiment, the invention provides a method of enhancingcardiac function in a subject, the method comprising administering apharmaceutical composition comprising a clonal population Jagged 1+and/or Frizzled 4+ human cardiac ventricular progenitor cells, such as aclonal population of at least at least 1×10⁹ cells, at least 2×10⁹cells, at least 5×10⁹ cells or at least 10×10⁹ cells. In one embodiment,the clonal population is administered directly into the heart of thesubject. For example, the clonal population can be administered directlyinto a ventricular region of the heart of the subject. In oneembodiment, the pharmaceutical composition administered to the subjectcomprises the clonal population formulated onto a three dimensionalmatrix, such as a heart muscle patch comprising ventricular musclecells. The subject is one in need of enhancement of cardiac function,for example someone who has suffered a myocardial infarction or someonewho has a congenital heart disorder.

In yet another aspect, the invention pertains to a method for generatinghuman ventricular tissue comprising

-   -   transplanting Jagged 1+ and/or Frizzled 4+ human cardiac        ventricular progenitor cells into an organ of a non-human        animal; and    -   allowing the progenitor cells to grow in vivo such that human        ventricular tissue is generated.        The non-human animal can be, for example, an immunodeficient        mouse. The organ can be, for example, the kidney (e.g., the        cells are transplanted under the kidney capsule) or the heart.

In one embodiment, the cells are transplanted at a time when one, two,three, four or five of the following cell marker patterns are present:(i) after peak of cardiac mesoderm formation; (ii) at time of peakIslet-1 expression; (iii) before peak of NKX2.5 expression; (iv) beforepeak expression of downstream genes MEF-2 and TBX-1; and (v) beforeexpression of differentiated contractile protein genes. In oneembodiment, the cells are transplanted between day 5 and day 7(inclusive) of in vitro culture of human pluripotent stem cells underconditions to generate human ventricular progenitor cells. In anotherembodiment, the cells are transplanted on day 6 of in vitro culture ofhuman pluripotent stem cells under conditions to generate humanventricular progenitor cells. The method can further include harvestingthe human ventricular tissue generated in the non-human animal.

In still another aspect of the invention, the human cardiac ventricularprogenitor cells described herein can be used in screening assays toevaluate the cardiac toxicity of a test compound. Accordingly, theinvention provides a method of screening for cardiac toxicity of testcompound, the method comprising

-   -   providing Jagged 1+ and/or Frizzled 4+ cardiac ventricular        progenitor cells;    -   contacting the cells with the test compound; and    -   measuring toxicity of the test compound for the cells,    -   wherein toxicity of the test compound for the cells indicates        cardiac toxicity of the test compound. The toxicity of the test        compound for the cells can be measured, for example, by        assessing cell viability or other physiological parameters of        the cell.

Culturing methods for generating human ventricular progenitor cells arealso provided. For example, in one embodiment, the invention pertains toa method of generating human ventricular progenitors (HVPs) comprising:

-   -   culturing human pluripotent stems cells (hPSCs) in a medium        comprising CHIR98014 such that cells expressing cardiac        mesodermal markers are generated, and    -   culturing the cells expressing cardiac mesodermal markers in a        medium comprising Wnt-C59 such that HVPs are generated.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of an exemplary culturing protocol forgenerating human Isl1+ cardiomyogenic progenitor cells from humanpluripotent stem cells (hPSCs).

FIG. 2 shows the results of Western blot analysis of protein expressionduring cardiac differentiation of hPSCs, showing expression of Isl1,Nkx2.5 and cTn1. GAPDH was used as a control.

FIG. 3 shows the results of flow cytometry analysis of cardiomyogenicprogenitor cells, showing expression of Isl1 on cells at day 6 ofdifferentiation.

FIG. 4 shows the results of double staining flow cytometry analysis ofcardiomyogenic progenitor cells, showing coexpression of Isl1 and Jag1on cells at day 6 of differentiation.

FIG. 5 shows the results of Western blot analysis of protein expressionduring cardiac differentiation of hPSCs, showing expression of FZD4.GAPDH was used as a control.

FIG. 6 shows the results of double staining flow cytometry analysis ofcardiomyogenic progenitor cells, showing coexpression of Isl1 and FZD4on cells at day 5 of differentiation.

FIG. 7 is a schematic diagram of the generation of human ventricularprogenitor (HVP) cells, their ultimate differentiation into ventricularmyocytes, their antibody purification and their use in transplantation.

FIGS. 8A-B is a schematic diagram of the transplantation of HPVs intothe renal capsule

(FIG. 8A) or intra-myocardially (FIG. 8B) for organ-on-organ tissueengineering.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods of isolating human cardiomyogenicprogenitor cells, in particular cells that are biased to the ventricularlineage, as well as isolated clonal populations of such progenitorcells, based on the discovery that Jagged 1 and Frizzled 4 are cellsurface markers for cardiac ventricular progenitor cells. In vitro andin vivo uses for these cardiac ventricular progenitor cells are alsoprovided.

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

As used herein, the terms “Jagged 1”, “Jag 1” and “JAG 1” are usedinterchangeably to refer to a protein known in the art that has beendescribed in, for example, Oda, T. et al. (1997) Genomics, 43:376-379;Oda, T. et al. (1997) Nat. Genet. 16:235-242; Li, L. et al. (1998)Immunity, 8:43-55; Bash, J. et al. (1999) EMBO J., 18:2803-2811; andJones, E. A. et al. (2000) J. Med. Genet. 37:658-662. A non-limitingexample of a Jagged 1 protein is the human protein having the amino acidsequence set forth in Genbank Accession Number P78504.3.

As used herein, the terms “Frizzled 4”, “Fzd 4” and “FZD 4” are usedinterchangeably to refer to a protein known in the art that has beendescribed in, for example, Kinkoshi, H. et al. (1999) Biochem. Biophys.Res. Commun., 264:955-961; Tanaka, S. et al. (1998) Proc. Natl. Acad.Sci. USA 95:10164-10169; and Robitaille, J. et al. (2002) Nat. Genet.,32:326-330. A non-limiting example of a Frizzled 4 protein is the humanprotein having the amino acid sequence set forth in Genbank AccessionNumber Q9ULV1.

As used herein, the term “stem cells” is used in a broad sense andincludes traditional stem cells, progenitor cells, pre-progenitor cells,reserve cells, and the like. The term “stem cell” or “progenitor” areused interchangeably herein, and refer to an undifferentiated cell whichis capable of proliferation and giving rise to more progenitor cellshaving the ability to generate a large number of mother cells that canin turn give rise to differentiated, or differentiable daughter cells.The daughter cells themselves can be induced to proliferate and produceprogeny that subsequently differentiate into one or more mature celltypes, while also retaining one or more cells with parentaldevelopmental potential. The term “stem cell” refers then, to a cellwith the capacity or potential, under particular circumstances, todifferentiate to a more specialized or differentiated phenotype, andwhich retains the capacity, under certain circumstances, to proliferatewithout substantially differentiating. In one embodiment, the termprogenitor or stem cell refers to a generalized mother cell whosedescendants (progeny) specialize, often in different directions, bydifferentiation, e.g., by acquiring completely individual characters, asoccurs in progressive diversification of embryonic cells and tissues.Cellular differentiation is a complex process typically occurringthrough many cell divisions. A differentiated cell may derive from amultipotent cell which itself is derived from a multipotent cell, and soon. While each of these multipotent cells may be considered stem cells,the range of cell types each can give rise to may vary considerably.Some differentiated cells also have the capacity to give rise to cellsof greater developmental potential. Such capacity may be natural or maybe induced artificially upon treatment with various factors. In manybiological instances, stem cells are also “multipotent” because they canproduce progeny of more than one distinct cell type, but this is notrequired for “stemness.” Self-renewal is the other classical part of thestem cell definition, and it is essential as used in this document. Intheory, self-renewal can occur by either of two major mechanisms. Stemcells may divide asymmetrically, with one daughter retaining the stemstate and the other daughter expressing some distinct other specificfunction and phenotype. Alternatively, some of the stem cells in apopulation can divide symmetrically into two stems, thus maintainingsome stem cells in the population as a whole, while other cells in thepopulation give rise to differentiated progeny only. Formally, it ispossible that cells that begin as stem cells might proceed toward adifferentiated phenotype, but then “reverse” and re-express the stemcell phenotype, a term often referred to as “dedifferentiation”.

The term “progenitor cell” is used herein to refer to cells that have acellular phenotype that is more primitive (e.g., is at an earlier stepalong a developmental pathway or progression than is a fullydifferentiated cell) relative to a cell which it can give rise to bydifferentiation. Often, progenitor cells also have significant or veryhigh proliferative potential. Progenitor cells can give rise to multipledistinct differentiated cell types or to a single differentiated celltype, depending on the developmental pathway and on the environment inwhich the cells develop and differentiate.

The term “pluripotent” as used herein refers to a cell with thecapacity, under different conditions, to differentiate to cell typescharacteristic of all three germ cell layers (endoderm, mesoderm andectoderm). Pluripotent cells are characterized primarily by theirability to differentiate to all three germ layers, using, for example, anude mouse and teratomas formation assay. Pluripotency is also evidencedby the expression of embryonic stem (ES) cell markers, although thepreferred test for pluripotency is the demonstration of the capacity todifferentiate into cells of each of the three germ layers. In someembodiments, a pluripotent cell is an undifferentiated cell.

The term “pluripotency” or a “pluripotent state” as used herein refersto a cell with the ability to differentiate into all three embryonicgerm layers: endoderm (gut tissue), mesoderm (including blood, muscle,and vessels), and ectoderm (such as skin and nerve), and typically hasthe potential to divide in vitro for a long period of time, e.g.,greater than one year or more than 30 passages.

The term “multipotent” when used in reference to a “multipotent cell”refers to a cell that is able to differentiate into some but not all ofthe cells derived from all three germ layers. Thus, a multipotent cellis a partially differentiated cell. Multipotent cells are well known inthe art, and examples of multipotent cells include adult stem cells,such as for example, hematopoietic stem cells and neural stem cells.Multipotent means a stem cell may form many types of cells in a givenlineage, but not cells of other lineages. For example, a multipotentblood stem cell can form the many different types of blood cells (red,white, platelets, etc.), but it cannot form neurons.

The term “embryonic stem cell” or “ES cell” or “ESC” are usedinterchangeably herein and refer to the pluripotent stem cells of theinner cell mass of the embryonic blastocyst (see U.S. Pat. Nos.5,843,780, 6,200,806, which are incorporated herein by reference). Suchcells can similarly be obtained from the inner cell mass of blastocystsderived from somatic cell nuclear transfer (see, for example, U.S. Pat.Nos. 5,945,577, 5,994,619, 6,235,970, which are incorporated herein byreference). The distinguishing characteristics of an embryonic stem celldefine an embryonic stem cell phenotype.

Accordingly, a cell has the phenotype of an embryonic stem cell if itpossesses one or more of the unique characteristics of an embryonic stemcell such that that cell can be distinguished from other cells.Exemplary distinguishing embryonic stem cell characteristics include,without limitation, gene expression profile, proliferative capacity,differentiation capacity, karyotype, responsiveness to particularculture conditions, and the like. In some embodiments, an ES cell can beobtained without destroying the embryo, for example, without destroyinga human embryo.

The term “adult stem cell” or “ASC” is used to refer to any multipotentstem cell derived from non-embryonic tissue, including fetal, juvenile,and adult tissue. Stem cells have been isolated from a wide variety ofadult tissues including blood, bone marrow, brain, olfactory epithelium,skin, pancreas, skeletal muscle, and cardiac muscle. Each of these stemcells can be characterized based on gene expression, factorresponsiveness, and morphology in culture. Exemplary adult stem cellsinclude neural stem cells, neural crest stem cells, mesenchymal stemcells, hematopoietic stem cells, and pancreatic stem cells. As indicatedabove, stem cells have been found resident in virtually every tissue.Accordingly, the present invention appreciates that stem cellpopulations can be isolated from virtually any animal tissue.

The term “human pluripotent stem cell” (abbreviated as hPSC), as usedherein, refers to a human cell that has the capacity to differentiateinto a variety of different cell types as discussed above regarding stemcells and pluripotency. Human pluripotent human stem cells include, forexample, induced pluripotent stem cells (iPSC) and human embryonic stemcells, such as ES cell lines.

The term “human cardiac progenitor cell”, as used herein, refers to ahuman progenitor cell that is committed to the cardiac lineage and thathas the capacity to differentiate into all three cardiac lineage cells(cardiac muscle cells, endothelial cells and smooth muscle cells).

The term “human cardiomyogenic progenitor cell”, as used herein, refersto a human progenitor cell that is committed to the cardiac lineage andthat predominantly differentiates into cardiac muscle cells (i.e., morethan 50% of the differentiated cells, preferably more than 60%, 70%, 80%or 90% of the differentiated cells, derived from the progenitor cellsare cardiac muscle cells).

The term “cardiac ventricular progenitor cell”, as used herein, refersto a progenitor cell that is committed to the cardiac lineage and thatpredominantly differentiates into cardiac ventricular muscle cells(i.e., more than 50% of the differentiated cells, preferably more than60%, 70%, 80% or 90% of the differentiated cells, derived from theprogenitor cells are cardiac ventricular muscle cells). This type ofcell is also referred to herein as a human ventricular progenitor, orHVP, cell.

The term “cardiomyocyte” refers to a muscle cell of the heart (e.g. acardiac muscle cell). A cardiomyocyte will generally express on its cellsurface and/or in the cytoplasm one or more cardiac-specific marker.Suitable cardiomyocyte-specific markers include, but are not limited to,cardiac troponin I, cardiac troponin-C, tropomyosin, caveolin-3, GATA-4,myosin heavy chain, myosin light chain-2a, myosin light chain-2v,ryanodine receptor, and atrial natriuretic factor.

The term “derived from” used in the context of a cell derived fromanother cell means that a cell has stemmed (e.g. changed from orproduced by) a cell which is a different cell type. The term “derivedfrom” also refers to cells which have been differentiated from aprogenitor cell.

The term “Isl1+ cardiac progenitor cell”, as used herein, refers to ahuman progenitor cell that is committed to the cardiac lineage and thatexpresses Islet 1.

The term “Isl1+ Jagged 1+ cardiac progenitor cell”, as used herein,refers to a human progenitor cell that is committed to the cardiaclineage and that expresses both Islet 1 and Jagged 1.

The term “Isl1+ Frizzled 4+ cardiac progenitor cell”, as used herein,refers to a human progenitor cell that is committed to the cardiaclineage and that expresses both Islet 1 and Frizzled 4.

The term “Isl1+ Jagged 1+ Frizzled 4+ cardiac progenitor cell”, as usedherein, refers to a human progenitor cell that is committed to thecardiac lineage and that expresses Islet 1, Jagged 1 and Frizzled 4.

With respect to cells in cell cultures or in cell populations, the term“substantially free Of” means that the specified cell type of which thecell culture or cell population is free, is present in an amount of lessthan about 10%, less than about 9%, less than about 8%, less than about7%, less than about 6%, less than about 5%, less than about 4%, lessthan about 3%, less than about 2% or less than about 1% of the totalnumber of cells present in the cell culture or cell population.

In the context of cell ontogeny, the adjective “differentiated”, or“differentiating” is a relative term. A “differentiated cell” is a cellthat has progressed further down the developmental pathway than the cellit is being compared with. Thus, stem cells can differentiate tolineage-restricted precursor cells (such as a mesodermal stem cell),which in turn can differentiate into other types of precursor cellsfurther down the pathway (such as an cardiomyocyte precursor), and thento an end-stage differentiated cell, which plays a characteristic rolein a certain tissue type, and may or may not retain the capacity toproliferate further.

The term “differentiation” in the present context means the formation ofcells expressing markers known to be associated with cells that are morespecialized and closer to becoming terminally differentiated cellsincapable of further differentiation. The pathway along which cellsprogress from a less committed cell, to a cell that is increasinglycommitted to a particular cell type, and eventually to a terminallydifferentiated cell is referred to as progressive differentiation orprogressive commitment. Cell which are more specialized (e.g., havebegun to progress along a path of progressive differentiation) but notyet terminally differentiated are referred to as partiallydifferentiated. Differentiation is a developmental process whereby cellsassume a specialized phenotype, e.g., acquire one or morecharacteristics or functions distinct from other cell types. In somecases, the differentiated phenotype refers to a cell phenotype that isat the mature endpoint in some developmental pathway (a so calledterminally differentiated cell). In many, but not all tissues, theprocess of differentiation is coupled with exit from the cell cycle. Inthese cases, the terminally differentiated cells lose or greatlyrestrict their capacity to proliferate. However, we note that in thecontext of this specification, the terms “differentiation” or“differentiated” refer to cells that are more specialized in their fateor function than at a previous point in their development, and includesboth cells that are terminally differentiated and cells that, althoughnot terminally differentiated, are more specialized than at a previouspoint in their development. The development of a cell from anuncommitted cell (for example, a stem cell), to a cell with anincreasing degree of commitment to a particular differentiated celltype, and finally to a terminally differentiated cell is known asprogressive differentiation or progressive commitment. A cell that is“differentiated” relative to a progenitor cell has one or morephenotypic differences relative to that progenitor cell. Phenotypicdifferences include, but are not limited to morphologic differences anddifferences in gene expression and biological activity, including notonly the presence or absence of an expressed marker, but alsodifferences in the amount of a marker and differences in theco-expression patterns of a set of markers.

The term “differentiation” as used herein refers to the cellulardevelopment of a cell from a primitive stage towards a more mature (i.e.less primitive) cell.

As used herein, “proliferating” and “proliferation” refers to anincrease in the number of cells in a population (growth) by means ofcell division. Cell proliferation is generally understood to result fromthe coordinated activation of multiple signal transduction pathways inresponse to the environment, including growth factors and othermitogens. Cell proliferation may also be promoted by release from theactions of intra- or extracellular signals and mechanisms that block ornegatively affect cell proliferation.

The terms “renewal” or “self-renewal” or “proliferation” are usedinterchangeably herein, and refers to a process of a cell making morecopies of itself (e.g. duplication) of the cell. In some embodiments,cells are capable of renewal of themselves by dividing into the sameundifferentiated cells (e.g. progenitor cell type) over long periods,and/or many months to years. In some instances, proliferation refers tothe expansion of cells by the repeated division of single cells into twoidentical daughter cells.

The term “lineages” as used herein refers to a term to describe cellswith a common ancestry or cells with a common developmental fate, forexample cells that have a developmental fate to develop into ventricularcardiomyocytes.

The term “clonal population”, as used herein, refers to a population ofcells that is derived from the outgrowth of a single cell. That is, thecells within the clonal population are all progeny of a single cell thatwas used to seed the clonal population.

The term “media” as referred to herein is a medium for maintaining atissue or cell population, or culturing a cell population (e.g. “culturemedia”) containing nutrients that maintain cell viability and supportproliferation. The cell culture medium may contain any of the followingin an appropriate combination: salt(s), buffer(s), amino acids, glucoseor other sugar(s), antibiotics, serum or serum replacement, and othercomponents such as peptide growth factors, etc. Cell culture mediaordinarily used for particular cell types are known to those skilled inthe art.

The term “phenotype” refers to one or a number of total biologicalcharacteristics that define the cell or organism under a particular setof environmental conditions and factors, regardless of the actualgenotype.

A “marker” as used herein describes the characteristics and/or phenotypeof a cell. Markers can be used for selection of cells comprisingcharacteristics of interest. Markers will vary with specific cells.Markers are characteristics, whether morphological, functional orbiochemical (enzymatic) characteristics particular to a cell type, ormolecules expressed by the cell type. Preferably, such markers areproteins, and more preferably, possess an epitope for antibodies orother binding molecules available in the art. However, a marker mayconsist of any molecule found in a cell including, but not limited to,proteins (peptides and polypeptides), lipids, polysaccharides, nucleicacids and steroids. Examples of morphological characteristics or traitsinclude, but are not limited to, shape, size, and nuclear to cytoplasmicratio. Examples of functional characteristics or traits include, but arenot limited to, the ability to adhere to particular substrates, abilityto incorporate or exclude particular dyes, ability to migrate underparticular conditions, and the ability to differentiate along particularlineages. Markers may be detected by any method available to one ofskill in the art.

The term “isolated cell” as used herein refers to a cell that has beenremoved from an organism in which it was originally found or adescendant of such a cell. Optionally the cell has been cultured invitro, e.g., in the presence of other cells. Optionally the cell islater introduced into a second organism or re-introduced into theorganism from which it (or the cell from which it is descended) wasisolated.

The term “isolated population” with respect to an isolated population ofcells as used herein refers to a population of cells that has beenremoved and separated from a mixed or heterogeneous population of cells.In some embodiments, an isolated population is a substantially purepopulation of cells as compared to the heterogeneous population fromwhich the cells were isolated or enriched from.

The term “substantially pure”, with respect to a particular cellpopulation, refers to a population of cells that is at least about 75%,preferably at least about 85%, more preferably at least about 90%, andmost preferably at least about 95% pure, with respect to the cellsmaking up a total cell population.

The terms “subject” and “individual” are used interchangeably herein,and refer to an animal, for example a human, to whom cardiac ventricularprogenitor cells as disclosed herein can be implanted into, for e.g.treatment, which in some embodiments encompasses prophylactic treatmentor for a disease model, with methods and compositions described herein,is or are provided. For treatment of disease states that are specificfor a specific animal such as a human subject, the term “subject” refersto that specific animal. The terms “non-human animals” and “non-humanmammals” are used interchangeably herein, and include mammals such asrats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-humanprimates. The term “subject” also encompasses any vertebrate includingbut not limited to mammals, reptiles, amphibians and fish. However,advantageously, the subject is a mammal such as a human, or othermammals such as a domesticated mammal, e.g. dog, cat, horse, and thelike, or production mammal, e.g. cow, sheep, pig, and the like are alsoencompassed in the term subject.

As used herein, the term “recipient” refers to a subject that willreceive a transplanted organ, tissue or cell.

The term “three-dimensional matrix” or “scaffold” or “matrices” as usedherein refers in the broad sense to a composition comprising abiocompatible matrix, scaffold, or the like. The three-dimensionalmatrix may be liquid, gel, semi-solid, or solid at 25° C. Thethree-dimensional matrix may be biodegradable or non-biodegradable. Insome embodiments, the three-dimensional matrix is biocompatible, orbioresorbable or bioreplacable. Exemplary three-dimensional matricesinclude polymers and hydrogels comprising collagen, fibrin, chitosan,MATRIGEL™, polyethylene glycol, dextrans including chemicallycrosslinkable or photocrosslinkable dextrans, processed tissue matrixsuch as submucosal tissue and the like. In certain embodiments, thethree-dimensional matrix comprises allogeneic components, autologouscomponents, or both allogeneic components and autologous components. Incertain embodiments, the three-dimensional matrix comprises synthetic orsemi-synthetic materials. In certain embodiments, the three-dimensionalmatrix comprises a framework or support, such as a fibrin-derivedscaffold.

As used herein, the terms “administering,” “introducing” and“transplanting” are used interchangeably and refer to the placement ofcardiomyogenic progenitor cells and/or cardiomyocytes differentiated asdescribed herein into a subject by a method or route which results in atleast partial localization of the cells at a desired site. The cells canbe administered by any appropriate route that results in delivery to adesired location in the subject where at least a portion of the cellsremain viable. The period of viability of the cells after administrationto a subject can be as short as a few hours, e.g. twenty-four hours, toa few days, to as long as several years.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) below normal, or lower, concentration of the marker. The termrefers to statistical evidence that there is a difference. It is definedas the probability of making a decision to reject the null hypothesiswhen the null hypothesis is actually true. The decision is often madeusing the p-value. The term “substantially” or “predominantly” as usedherein means a proportion of at least about 60%, or preferably at leastabout 70% or at least about 80%, or at least about 90%, at least about95%, at least about 97% or at least about 99% or more, or any integerbetween 70% and 100%.

The term “disease” or “disorder” is used interchangeably herein, andrefers to any alternation in state of the body or of some of the organs,interrupting or disturbing the performance of the functions and/orcausing symptoms such as discomfort, dysfunction, distress, or evendeath to the person afflicted or those in contact with a person. Adisease or disorder can also related to a distemper, ailing, ailment,malady, disorder, sickness, illness, complaint, indisposition oraffection.

As used herein, the phrase “cardiovascular condition, disease ordisorder” is intended to include all disorders characterized byinsufficient, undesired or abnormal cardiac function, e.g. ischemicheart disease, hypertensive heart disease and pulmonary hypertensiveheart disease, valvular disease, congenital heart disease and anycondition which leads to congestive heart failure in a subject,particularly a human subject. Insufficient or abnormal cardiac functioncan be the result of disease, injury and/or aging. By way of background,a response to myocardial injury follows a well-defined path in whichsome cells die while others enter a state of hibernation where they arenot yet dead but are dysfunctional. This is followed by infiltration ofinflammatory cells, deposition of collagen as part of scarring, all ofwhich happen in parallel with in-growth of new blood vessels and adegree of continued cell death. As used herein, the term “ischemia”refers to any localized tissue ischemia due to reduction of the inflowof blood. The term “myocardial ischemia” refers to circulatorydisturbances caused by coronary atherosclerosis and/or inadequate oxygensupply to the myocardium. For example, an acute myocardial infarctionrepresents an irreversible ischemic insult to myocardial tissue. Thisinsult results in an occlusive (e.g., thrombotic or embolic) event inthe coronary circulation and produces an environment in which themyocardial metabolic demands exceed the supply of oxygen to themyocardial tissue.

As used herein, the term “treating” or “treatment” are usedinterchangeably herein and refers to reducing or decreasing oralleviating or halting at least one adverse effect or symptom of acardiovascular condition, disease or disorder, i.e., any disordercharacterized by insufficient or undesired cardiac function. Adverseeffects or symptoms of cardiac disorders are well-known in the art andinclude, but are not limited to, dyspnea, chest pain, palpitations,dizziness, syncope, edema, cyanosis, pallor, fatigue and death. In someembodiments, the term “treatment” as used herein refers to prophylactictreatment or preventative treatment to prevent the development of asymptom of a cardiovascular condition in a subject.

Treatment is generally “effective” if one or more symptoms or clinicalmarkers are reduced as that term is defined herein. Alternatively, atreatment is “effective” if the progression of a disease is reduced orhalted. That is, “treatment” includes not just the improvement ofsymptoms or decrease of markers of the disease, but also a cessation orslowing of progress or worsening of a symptom that would be expected inabsence of treatment. Beneficial or desired clinical results include,but are not limited to, alleviation of one or more symptom(s),diminishment of extent of disease, stabilized (i.e., not worsening)state of disease, delay or slowing of disease progression, ameliorationor palliation of the disease state, and remission (whether partial ortotal), whether detectable or undetectable. “Treatment” can also meanprolonging survival as compared to expected survival if not receivingtreatment. Those in need of treatment include those already diagnosedwith a cardiac condition, as well as those likely to develop a cardiaccondition due to genetic susceptibility or other factors such as weight,diet and health. In some embodiments, the term to treat also encompassespreventative measures and/or prophylactic treatment, which includesadministering a pharmaceutical composition as disclosed herein toprevent the onset of a disease or disorder.

A therapeutically significant reduction in a symptom is, e.g. at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 100%, at least about 125%,at least about 150% or more in a measured parameter as compared to acontrol or non-treated subject. Measured or measurable parametersinclude clinically detectable markers of disease, for example, elevatedor depressed levels of a biological marker, as well as parametersrelated to a clinically accepted scale of symptoms or markers for adisease or disorder. It will be understood, that the total daily usageof the compositions and formulations as disclosed herein will be decidedby the attending physician within the scope of sound medical judgment.The exact amount required will vary depending on factors such as thetype of disease being treated.

With reference to the treatment of a cardiovascular condition or diseasein a subject, the term “therapeutically effective amount” refers to theamount that is safe and sufficient to prevent or delay the developmentor a cardiovascular disease or disorder. The amount can thus cure orcause the cardiovascular disease or disorder to go into remission, slowthe course of cardiovascular disease progression, slow or inhibit asymptom of a cardiovascular disease or disorder, slow or inhibit theestablishment of secondary symptoms of a cardiovascular disease ordisorder or inhibit the development of a secondary symptom of acardiovascular disease or disorder. The effective amount for thetreatment of the cardiovascular disease or disorder depends on the typeof cardiovascular disease to be treated, the severity of the symptoms,the subject being treated, the age and general condition of the subject,the mode of administration and so forth. Thus, it is not possible tospecify the exact “effective amount”. However, for any given case, anappropriate “effective amount” can be determined by one of ordinaryskill in the art using only routine experimentation. The efficacy oftreatment can be judged by an ordinarily skilled practitioner, forexample, efficacy can be assessed in animal models of a cardiovasculardisease or disorder as discussed herein, for example treatment of arodent with acute myocardial infarction or ischemia-reperfusion injury,and any treatment or administration of the compositions or formulationsthat leads to a decrease of at least one symptom of the cardiovasculardisease or disorder as disclosed herein, for example, increased heartejection fraction, decreased rate of heart failure, decreased infarctsize, decreased associated morbidity (pulmonary edema, renal failure,arrhythmias) improved exercise tolerance or other quality of lifemeasures, and decreased mortality indicates effective treatment. Inembodiments where the compositions are used for the treatment of acardiovascular disease or disorder, the efficacy of the composition canbe judged using an experimental animal model of cardiovascular disease,e.g., animal models of ischemia-reperfusion injury (Headrick J P, Am JPhysiol Heart circ Physiol 285; H1797; 2003) and animal models acutemyocardial infarction. (Yang Z, Am J Physiol Heart Circ. Physiol282:H949:2002; Guo Y, J Mol Cell Cardiol 33; 825-830, 2001). When usingan experimental animal model, efficacy of treatment is evidenced when areduction in a symptom of the cardiovascular disease or disorder, forexample, a reduction in one or more symptom of dyspnea, chest pain,palpitations, dizziness, syncope, edema, cyanosis, pallor, fatigue andhigh blood pressure which occurs earlier in treated, versus untreatedanimals. By “earlier” is meant that a decrease, for example in the sizeof the tumor occurs at least 5% earlier, but preferably more, e.g., oneday earlier, two days earlier, 3 days earlier, or more.

As used herein, the term “treating” when used in reference to atreatment of a cardiovascular disease or disorder is used to refer tothe reduction of a symptom and/or a biochemical marker of acardiovascular disease or disorder, for example a reduction in at leastone biochemical marker of a cardiovascular disease by at least about 10%would be considered an effective treatment. Examples of such biochemicalmarkers of cardiovascular disease include a reduction of, for example,creatine phosphokinase (CPK), aspartate aminotransferase (AST), lactatedehydrogenase (LDH) in the blood, and/or a decrease in a symptom ofcardiovascular disease and/or an improvement in blood flow and cardiacfunction as determined by someone of ordinary skill in the art asmeasured by electrocardiogram (ECG or EKG), or echocardiogram (heartultrasound), Doppler ultrasound and nuclear medicine imaging. Areduction in a symptom of a cardiovascular disease by at least about 10%would also be considered effective treatment by the methods as disclosedherein. As alternative examples, a reduction in a symptom ofcardiovascular disease, for example a reduction of at least one of thefollowing; dyspnea, chest pain, palpitations, dizziness, syncope, edema,cyanosis etc. by at least about 10% or a cessation of such systems, or areduction in the size one such symptom of a cardiovascular disease by atleast about 10% would also be considered as affective treatments by themethods as disclosed herein. In some embodiments, it is preferred, butnot required that the therapeutic agent actually eliminate thecardiovascular disease or disorder, rather just reduce a symptom to amanageable extent.

Subjects amenable to treatment by the methods as disclosed herein can beidentified by any method to diagnose myocardial infarction (commonlyreferred to as a heart attack) commonly known by persons of ordinaryskill in the art are amenable to treatment using the methods asdisclosed herein, and such diagnostic methods include, for example butare not limited to; (i) blood tests to detect levels of creatinephosphokinase (CPK), aspartate aminotransferase (AST), lactatedehydrogenase (LDH) and other enzymes released during myocardialinfarction; (ii) electrocardiogram (ECG or EKG) which is a graphicrecordation of cardiac activity, either on paper or a computer monitor.An ECG can be beneficial in detecting disease and/or damage; (iii)echocardiogram (heart ultrasound) used to investigate congenital heartdisease and assessing abnormalities of the heart wall, includingfunctional abnormalities of the heart wall, valves and blood vessels;(iv) Doppler ultrasound can be used to measure blood flow across a heartvalve; (v) nuclear medicine imaging (also referred to as radionuclidescanning in the art) allows visualization of the anatomy and function ofan organ, and can be used to detect coronary artery disease, myocardialinfarction, valve disease, heart transplant rejection, check theeffectiveness of bypass surgery, or to select patients for angioplastyor coronary bypass graft.

The terms “coronary artery disease” and “acute coronary syndrome” asused interchangeably herein, and refer to myocardial infarction refer toa cardiovascular condition, disease or disorder, include all disorderscharacterized by insufficient, undesired or abnormal cardiac function,e.g. ischemic heart disease, hypertensive heart disease and pulmonaryhypertensive heart disease, valvular disease, congenital heart diseaseand any condition which leads to congestive heart failure in a subject,particularly a human subject. Insufficient or abnormal cardiac functioncan be the result of disease, injury and/or aging. By way of background,a response to myocardial injury follows a well-defined path in whichsome cells die while others enter a state of hibernation where they arenot yet dead but are dysfunctional. This is followed by infiltration ofinflammatory cells, deposition of collagen as part of scarring, all ofwhich happen in parallel with in-growth of new blood vessels and adegree of continued cell death.

As used herein, the term “ischemia” refers to any localized tissueischemia due to reduction of the inflow of blood. The term “myocardialischemia” refers to circulatory disturbances caused by coronaryatherosclerosis and/or inadequate oxygen supply to the myocardium. Forexample, an acute myocardial infarction represents an irreversibleischemic insult to myocardial tissue. This insult results in anocclusive (e.g., thrombotic or embolic) event in the coronarycirculation and produces an environment in which the myocardialmetabolic demands exceed the supply of oxygen to the myocardial tissue.

The terms “composition” or “pharmaceutical composition” usedinterchangeably herein refer to compositions or formulations thatusually comprise an excipient, such as a pharmaceutically acceptablecarrier that is conventional in the art and that is suitable foradministration to mammals, and preferably humans or human cells. In someembodiments, pharmaceutical compositions can be specifically formulatedfor direct delivery to a target tissue or organ, for example, by directinjection or via catheter injection to a target tissue. In otherembodiments, compositions can be specifically formulated foradministration via one or more of a number of routes, including but notlimited to, oral, ocular parenteral, intravenous, intraarterial,subcutaneous, intranasal, sublingual, intraspinal,intracerebroventricular, and the like. In addition, compositions fortopical (e.g., oral mucosa, respiratory mucosa) and/or oraladministration can form solutions, suspensions, tablets, pills,capsules, sustained-release formulations, oral rinses, or powders, asknown in the art are described herein. The compositions also can includestabilizers and preservatives. For examples of carriers, stabilizers andadjuvants, University of the Sciences in Philadelphia (2005) Remington:The Science and Practice of Pharmacy with Facts and Comparisons, 21stEd.

As used herein, the terms “administering,” “introducing” and“transplanting” are used interchangeably and refer to the placement of apharmaceutical composition comprising cardiomyogenic progenitor cells,or a composition comprising a population of differentiated cardiomyoctes(e.g., ventricular cardiomyocytes) as described herein, into a subjectby a method or route which results in at least partial localization ofthe pharmaceutical composition, at a desired site or tissue location. Insome embodiments, the pharmaceutical composition can be administered byany appropriate route which results in effective treatment in thesubject, i.e. administration results in delivery to a desired locationor tissue in the subject where at least a portion of the cells arelocated at a desired target tissue or target cell location.

The phrases “parenteral administration” and “administered parenterally”as used herein mean modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intraventricular, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, sub capsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. The phrases “systemicadministration,” “administered systemically”, “peripheraladministration” and “administered peripherally” as used herein mean theadministration of cardiovascular stem cells and/or their progeny and/orcompound and/or other material other than directly into the cardiactissue, such that it enters the animal's system and, thus, is subject tometabolism and other like processes, for example, subcutaneous orintravenous administration.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject agents fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation.

The term “drug” or “compound” or “test compound” as used herein refersto a chemical entity or biological product, or combination of chemicalentities or biological products, administered to a subject to treat orprevent or control a disease or condition. The chemical entity orbiological product is preferably, but not necessarily a low molecularweight compound, but may also be a larger compound, for example, anoligomer of nucleic acids, amino acids, or carbohydrates includingwithout limitation proteins, oligonucleotides, ribozymes, DNAzymes,glycoproteins, siRNAs, lipoproteins, aptamers, and modifications andcombinations thereof.

The term “transplantation” as used herein refers to introduction of newcells (e.g. reprogrammed cells), tissues (such as differentiated cellsproduced from reprogrammed cells), or organs into a host (i.e.transplant recipient or transplant subject)

The term “agent reactive with Jagged 1”, as used herein, refers to anagent that binds to or otherwise interacts with Jagged 1. Preferably,the agent “specifically” binds or otherwise interacts with Jagged 1 suchthat it does not bind or interact with other non-Jagged 1 proteins.

The term “agent reactive with Frizzled 4”, as used herein, refers to anagent that binds to or otherwise interacts with Frizzled 4. Preferably,the agent “specifically” binds or otherwise interacts with Frizzled 4such that it does not bind or interact with other non-Frizzled 4proteins.

The term “agent reactive with Islet 1”, as used herein, refers to anagent that binds to or otherwise interacts with Islet 1. Preferably, theagent “specifically” binds or otherwise interacts with Islet 1 such thatit does not bind or interact with other non-Islet 1 proteins.

The term “antibody”, as used herein, includes whole antibodies and anyantigen binding fragment (i.e., “antigen-binding portion”) or singlechain thereof. An “antibody” refers, in one preferred embodiment, to aglycoprotein comprising at least two heavy (H) chains and two light (L)chains inter-connected by disulfide bonds, or an antigen binding portionthereof. Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as V_(H)) and a heavy chain constant region. Theheavy chain constant region is comprised of three domains, CH1, CH2 andCH3. Each light chain is comprised of a light chain variable region(abbreviated herein as V_(L)) and a light chain constant region. Thelight chain constant region is comprised of one domain, CL. The term“antigen-binding portion” of an antibody (or simply “antibody portion”),as used herein, refers to one or more fragments of an antibody thatretain the ability to specifically bind to an antigen.

The term “monoclonal antibody,” as used herein, refers to an antibodythat displays a single binding specificity and affinity for a particularepitope.

The term “human monoclonal antibody”, as used herein, refers to anantibody which displays a single binding specificity and which hasvariable and optional constant regions derived from human germlineimmunoglobulin sequences.

The term “humanized monoclonal antibody”, as used herein, refers to anantibody which displays a single binding specificity and which has heavyand light chain CDR1, 2 and 3 from a non-human antibody (e.g., a mousemonoclonal antibody) grafted into human framework and constant regions.

The term “chimeric monoclonal antibody”, as used herein, refers to anantibody which displays a single binding specificity and which has heavyand light chain variable regions from one species linked to constantregions from another species.

The term “fusion protein”, as used herein, refers to a compositeprotein, typically made using recombinant DNA technology, in which twodifferent proteins, or portions thereof, are operatively linkedtogether. A non-limiting example is an Fc fusion protein in which anon-immunoglobulin protein is operatively linked to an immunoglobulin Fcregion.

Various aspects of the invention are described in further detail in thefollowing subsections.

Methods of Isolating Human Cardiac Ventricular Progenitor Cells

In one aspect, the invention pertains to methods of isolating humancardiac ventricular progenitor cells. As described in the Examples,Jagged 1 and Frizzled 4 have now been identified as a cell surfacemarker of human cardiac ventricular progenitor cells and thus thismarker can be used to facilitate isolation of these progenitor cells.Accordingly, in one embodiment, the invention provides a method forisolating human cardiac ventricular progenitor cells, the methodcomprising:

-   -   contacting a culture of human cells containing cardiac        progenitor cells with one or more agents reactive with Jagged 1        and/or Frizzled 4; and    -   separating Jagged 1 and/or Frizzled 4 reactive positive cells        from non-reactive cells to thereby isolate human cardiac        ventricular progenitor cells.

Alternatively, after the contacting step, the method can compriseisolating Jagged 1 and/or Frizzled 4 reactive positive cells fromnon-reactive cells to thereby isolate human cardiac ventricularprogenitor cells.

Also as described in the Examples, Islet 1 is a marker that isco-expressed with Jagged 1 and/or Frizzled 4 by the cardiac ventricularprogenitor cells and thus both markers can be used to facilitateisolation of these progenitor cells. Accordingly, in another embodimentof the above method, the culture of human cells is also contacted withan agent reactive with Islet 1; and Jagged 1 reactive/Islet 1 reactivepositive cells, Frizzled 4 reactive/Islet 1 reactive positive cells orJagged 1 reactive/Frizzled 4 reactive/Islet 1 reactive positive cellsare separated from non-reactive cells to thereby isolate human cardiacventricular progenitor cells. The culture of human cells can besimultaneously contacted with the agent(s) reactive with Jagged 1 and/orFrizzled 4 and the agent reactive with Islet 1. Alternatively, theculture of human cells can be contacted with the agent reactive withIslet 1 before contacting with the agent(s) reactive with Jagged 1and/or Frizzled 4. Alternatively, the culture of human cells can becontacted with the agent(s) reactive with Jagged 1 and/or Frizzled 4before contacting with the agent reactive with Islet 1.

In yet another embodiment, the culture of human cells can be contactedwith an agent reactive with Jagged 1 and an agent reactive with Frizzled4, and cells that are Jagged 1+/Frizzled 4+ can be separated. In yetanother embodiment, the culture of human cells can be contacted with anagent reactive with Jagged 1, an agent reactive with Frizzled 4 and anagent reactive with Islet 1, and cells that are Jagged 1+/Frizzled4+/Islet 1+ can be separated.

In another embodiment, the invention provides a method for isolatinghuman cardiac ventricular progenitor cells, the method comprising:

-   -   culturing human pluripotent stem cells under conditions that        generate cardiac progenitor cells to obtain a culture of cells;    -   contacting the culture of cells with one or more agents reactive        with Jagged 1 and/or Frizzled 4; and    -   separating Jagged 1 reactive positive cells and/or Frizzled 4        reactive positive cells from non -reactive cells to thereby        isolate human cardiac ventricular progenitor cells.

Alternatively, after the culturing and contacting steps, the method cancomprise isolating Jagged 1 and/or Frizzled 4 reactive positive cellsfrom non-reactive cells to thereby isolate human cardiac ventricularprogenitor cells.

In one embodiment of the above method, the culture of human cells isalso contacted with an agent reactive with Islet 1; and Jagged 1reactive/Islet 1 reactive, Frizzled 4 reactive/Islet 1 reactive positivecells or Jagged 1 reactive/Frizzled 4 reactive/Islet 1 reactive positivecells are separated from non-reactive cells to thereby isolate humancardiac ventricular progenitor cells. The culture of human cells can besimultaneously contacted with the agent(s) reactive with Jagged 1 and/orFrizzled 4 and the agent(s) reactive with Islet 1. Alternatively, theculture of human cells can be contacted with the agent(s) reactive withIslet 1 before contacting with the agent reactive with Jagged 1 and/orFrizzled 4. Alternatively, the culture of human cells can be contactedwith the agent(s) reactive with Jagged 1 and/or Frizzled 4 beforecontacting with the agent reactive with Islet 1.

In yet another embodiment, the culture of human cells can be contactedwith an agent reactive with Jagged 1 and an agent reactive with Frizzled4, and cells that are Jagged 1+/Frizzled 4+ can be separated. In yetanother embodiment, the culture of human cells can be contacted with anagent reactive with Jagged 1, an agent reactive with Frizzled 4 and anagent reactive with Islet 1, and cells that are Jagged 1+/Frizzled4+/Islet 1+ can be separated.

In a preferred embodiment, the agent reactive with Jagged 1 is ananti-Jagged 1 antibody, such as a monoclonal antibody. Non-limitingexamples include murine, rabbit, human, humanized or chimeric monoclonalantibodies with binding specificity for Jagged 1. Anti-Jagged 1monoclonal antibodies are commercially available in the art (e.g., R&DSystems, Santa Cruz Biotechnology). Moreover, anti-Jagged 1 antibodiescan be prepared using standard techniques well established in the artusing Jagged 1 as the antigen.

In another embodiment, the agent reactive with Jagged 1 is a Jagged 1ligand, such as a soluble Jagged 1 ligand or a soluble Jagged 1 ligandfusion protein. Non-limiting examples of Jagged 1 ligands includeNotch-1 and Notch-2. Preferably, the Jagged 1 ligand is Notch-1. SolubleJagged 1 ligands, such as a soluble form of Notch-1, can be preparedusing standard recombinant DNA techniques, for example by deletion ofthe transmembrane and cytoplasmic domains. A soluble ligand can betransformed into a soluble ligand fusion protein also using standardrecombinant DNA techniques. A fusion protein can be prepared in whichfusion partner can comprise a binding moiety that facilitates separationof the fusion protein.

Similarly, the agent reactive with Frizzled 4 can be, for example, ananti-Frizzled 4 antibody (e.g., monoclonal antibody) or a Frizzled 4ligand, such as a Frizzled 4 ligand fusion protein. Non-limitingexamples include murine, rabbit, human, humanized or chimeric monoclonalantibodies with binding specificity for Frizzled 4. Anti-Frizzled 4monoclonal antibodies are commercially available in the art (e.g., R&DSystems, Santa Cruz Biotechnology). Moreover, anti-Frizzled 4 antibodiescan be prepared using standard techniques well established in the artusing Frizzled 4 as the antigen. Non-limiting examples of Frizzled 4ligands include Nestin proteins and Wnt receptors.

Similarly, the agent reactive with Islet 1 can be, for example, ananti-Islet 1 antibody (e.g., monoclonal antibody) or an Islet 1 ligand,such as an Islet 1 ligand fusion protein.

In order to separate the Jagged 1 and/or Frizzled 4 reactive positivecells from non-reactive cells, one of a variety of different cellseparation techniques known in the art can be used. Preferably, theJagged 1 and/or Frizzled 4 reactive positive cells are separated fromnon-reactive cells by fluorescence activated cell sorting (FACS). TheFACS technology, and apparatuses for carrying it out to separate cells,is well established in the art. When FACS is used for cell separation,preferably the agent(s) reactive with Jagged 1 and/or Frizzled 4 that isused is a fluorescently-labeled anti-Jagged 1 and/or anti-Frizzled 4monoclonal antibody. Alternatively, cell separation can be achieved by,for example, magnetic activated cell sorting (MACS). When MACS is usedfor cell separation, preferably the agent reactive with Jagged 1 orFrizzled 4 that is used is magnetic nanoparticles coated withanti-Jagged 1 or anti-Frizzled 4 monoclonal antibody. Alternatively,other single cell sorting methodologies known in the art can be appliedto the methods of isolating cardiac ventricular progenitor cells of theinvention, including but not limited to IsoRaft array and DEPArraytechnologies.

Prior to contact with the agent(s) reactive with Jagged 1 and/orFrizzled 4, and separation of Jagged 1 and/or Frizzled 4 reactive cells,human pluripotent stem cells can be cultured under conditions that leadto the generation of cardiac progenitor cells. Culture conditions forgenerating cardiac progenitor cells have been described in the art (seee.g., Lian, X. et al. (2012) Proc. Natl. Acad. Sci. USA 109:E1848-1857;U.S. Patent Publication No. 20130189785) and also are described indetail in Example 1 and FIG. 1, as well as in Example 10. Typically,Wnt/β-catenin signaling is first activated in the hPSCs, followed by anincubation period, followed by inhibition of Wnt/β-catenin signaling.Activation of Wnt/β-catenin signaling is achieved by incubation with aGsk3 inhibitor, preferably CHIR98014 (CAS 556813-39-9). Inhibition ofWnt/β-catenin signaling is achieved by incubation with a Porcninhibitor, preferably Wnt-C59 (CAS 1243243-89-1). Suitable hPSCs for usein the methods of the invention include induced pluripotent stem cells(iPSC), such as 19-11-1, 19-9-7 or 6-9-9 cells (Yu, J. et al. (2009)Science 324:797-801), and human embryonic stem cell lines, such as ES03cells (WiCell Research Institute) or H9 cells (Thomson, J. A. et al.(1998) Science 282:1145-1147). Suitable culture media for generatingcardiomyogenic progenitors include E8 medium, mTeSR1 medium and RPMI/B27minus insulin, each described further in Example 1 and/or Example 10.

Preferably, the human cardiomyogenic progenitor cells are ventricularprogenitor cells. Culture conditions have now been determined that biasthe cardiomyogenic progenitor cells to the ventricular lineage. Theseventricular cardiomyogenic progenitor cells can be cultured in RPMI/B27medium and they can further differentiate into ventricular muscle cells.A preferred medium for culturing the cardiac ventricular progenitorcells in vitro such that they differentiation into ventricular cells invitro (e.g., expressing the MLC2v marker described below) is the CardiacProgenitor Culture (CPC) medium (advanced DMEM/F12 supplemented with 20%KnockOut Serum Replacement, 2.5 mM GlutaMAX and 100 μg/ml Vitamin C).

Known markers of differentiated cardiac cells can be used to identifythe type(s) of cells that are generated by differentiation of thecardiac progenitor cells. For example, cardiac troponin I (cTnI) can beused as a marker of cardiomyocyte differentiation. CD144 (VE-cadherin)can be used as a marker of endothelial cells. Smooth muscle actin (SMA)can be used as a marker of smooth muscle cells. MLC2v can be used as amarker of ventricular muscle cells. MLC2a, which is expressed on bothimmature ventricular muscle cells and atrial muscle cells, can be usedas a marker for those cell types. Additionally, sarcolipin, which isspecifically expressed in atrial muscle cells, can be used as a markerfor atrial muscle cells. Phospholamban, which is expressed predominantlyin the ventricles and, to a lesser extent, in the atria, can also beused as a marker. Hairy-related transcription factor 1 (HRT1), alsocalled Hey1, which is expressed in atrial cardiomyocytes, can be used asa marker for atrial cardiomyocytes. HRT2 (Hey2), which is expressed inventricular cardiomyocytes, can be used as a marker for ventricularcardiomyocytes. In addition, IRX4 has a ventricular-restrictedexpression pattern during all stages of development, and thus can beused as a ventricular lineage marker. In summary, the genes expressed inthe ventricles, and thus which are appropriate ventricular markers, are:MLC2v, IRX4 and HRT2, while genes expressed in the atria, and thus whichare appropriate atrial markers are: MLC2a, HRT1, Sarcolipin and ANF(atrial natriuretic factor). The preferred marker of ventriculardifferentiation is MLC2v.

Clonal Populations of Human Cardiac Ventricular Progenitor Cells

In another aspect, the invention provides methods for obtaining a clonalpopulation of human cardiac ventricular progenitor cells, as well asisolated clonal populations of such progenitors. The invention allowsfor the expansion and propagation of the cardiac ventricular progenitorcells such that a clonal population of a billion or more cells can beachieved. The ability to clonally expand the Jagged 1+ and/or Frizzled4+ cardiac ventricular progenitor cells to such large numbers is anecessary feature for successful use of these cells in vivo to enhancecardiac function, since such a use requires on the order of a billion ormore cells.

Accordingly, in another aspect, the invention provides a method forobtaining a clonal population of human cardiac ventricular progenitorcells, the method comprising:

isolating a single Jagged 1+ and/or Frizzled 4+ human cardiacventricular progenitor cell; and

culturing the single Jagged 1+ and/or Frizzled 4+ human cardiacventricular progenitor cell under conditions such that the cell isexpanded to at least 1×10⁹ cells to thereby obtain a clonal populationof human cardiac ventricular progenitor cells.

In a preferred embodiment, the single Jagged 1+ and/or Frizzled 4+ humancardiac ventricular progenitor cell is Islet 1 positive, Nkx2.5 negativeand flk1 negative at the time of initial culture. As described furtherin the Examples, such a single cell can be obtained at approximately day6 of the culture under conditions that promote the generation ofcardiomyogenic progenitors. The clonal population of human cardiacventricular progenitors can be further cultured and differentiated invitro such that the cells express the ventricular maker MLC2v.

Preferably, the single Jagged 1+ and/or Frizzled 4+ human cardiacventricular progenitor cell is isolated by fluorescence activated cellsorting. Alternatively, the cell can be isolated by MACS or by othercell sorting methods known in the art and/or described herein.

Preferably, the single Jagged 1+ and/or Frizzled 4+ human cardiacventricular progenitor cell is isolated using one or more agentsreactive with Jagged 1 and/or Frizzled 4, such as an anti-Jagged 1antibody or other agent reactive with Jagged 1 as describedhereinbefore, or an anti-Frizzled 4 antibody or other agent reactivewith Frizzled 4 as described hereinbefore.

In other embodiments, the clonal population of human cardiac ventricularprogenitor cells is Jagged 1+ and Frazzled 4+. Such double-positivecells can be isolated and clonally expanded as described herein beforeusing both an agent reactive with Jagged 1 and an agent reactive withFrazzled 4.

In a preferred embodiment, the single Jagged 1+ and/or Frizzled 4+ humancardiac ventricular progenitor cell is cultured in Cardiac ProgenitorCulture (CPC) medium, as described hereinbefore

In a preferred embodiment, the single Jagged 1+ and/or Frizzled 4+ humancardiac ventricular progenitor cell is cultured under conditions suchthat the cell is biased toward ventricular differentiation. Preferredculture conditions include culture in CPC medium.

In various embodiments, the single Jagged 1+ and/or Frizzled 4+ humancardiac ventricular progenitor cell can be expanded to at least 1×10⁹cells, at least 2×10⁹ cells, at least 3×10⁹ cells, at least 4×10⁹ cells,at least 5×10⁹ cells, at least 6×10⁹ cells, at least 7×10⁹ cells, atleast 8×10⁹ cells, at least 9×10⁹ cells or at least 10×10⁹ cells.

Accordingly, the invention also provides a clonal population of at least1×10⁹ Jagged 1+ and/or Frizzled 4+ human cardiac ventricular progenitorcells, which are obtainable or obtained by the methods of the inventionfor obtaining a clonal population of human cardiac ventricularprogenitor cells. In various embodiments, the clonal population ofJagged 1+ and/or Frizzled 4+ human cardiac ventricular progenitor cellscomprises at least 1×10⁹ cells, at least 2×10⁹ cells, at least 3×10⁹cells, at least 4×10⁹ cells, at least 5×10⁹ cells, at least 6×10⁹ cells,at least 7×10⁹ cells, at least 8×10⁹ cells, at least 9×10⁹ cells or atleast 10×10⁹ cells. Differentiation of the progenitor cells to theventricular lineage in vitro can be achieved by culture under conditionsdescribed herein for biasing toward the ventricular lineage.Furthermore, transplantation of the cardiac ventricular progenitor cellsin vivo leads to ventricular differentiation in vivo.

The invention also provides pharmaceutical compositions comprising theclonal population of cardiac ventricular progenitor cells. Thepharmaceutical compositions typically are sterile and can comprisebuffers, media, excipients and the like suitable for pharmaceuticaladministration. In one embodiment, the pharmaceutical compositioncomprising the clonal population is formulated onto a three dimensional(3D) matrix. Compositions formulated onto a 3D matrix are particularlypreferred for formation of a heart muscle cell patch that can betransplanted in vivo for heart muscle repair. Furthermore, thecompositions can be formulated into two dimensional (2D) sheets ofcells, such as a muscular thin film (MTF) as described in Domian, I. J.et al. (2009) Science 326:426-429. Such 2D sheets of cell tissue alsocan be used in the formation of a heart muscle cell patch that can betransplanted in vivo for heart muscle repair.

Generation of Human Ventricular Progenitors (HVPs)

Prior to isolation by the aforementioned methods, and optionallyobtaining a clonal population by the aforementioned methods, anon-clonal population of human ventricular progenitors (HVPs) can beobtained by culture of human pluripotent stem cells (hPSCs) underappropriate culture conditions to generate the HVPs. An exemplary set ofculture conditions, and suitable starting cells, is described in detailin Example 1 and Example 10, also referred to herein as the HumanVentricular Progenitor Generation (HVPG) protocol. Suitable hPSCstarting cells include induced pluripotent stem cells (iPSC) and humanembryonic stem cells, such as ES cell lines. For the protocol,Wnt/β-catenin signaling first is activated in the hPSCs, followed by anincubation period, followed by inhibition of Wnt/β-catenin signaling.Wnt/β-catenin signaling activation is achieved by incubation with a Gsk3inhibitor, preferably CHIR98014 (CAS 556813-39-9; commercially availablefrom, e.g., Selleckchem). Wnt/β-catenin signaling inhibition is achievedby incubation with a Porcn inhibitor, preferably Wnt-C59 (CAS1243243-89-1; commercially available from, e.g., Selleckchem or Tocris).The Gsk3 inhibitor is used to promote cardiac mesodermaldifferentiation, whereas the Porcn inhibitor is used to enhanceventricular progenitor differentiation from mesoderm cells.

Accordingly, in another aspect, the invention provides a method ofgenerating human ventricular progenitors (HVPs) comprising culturinghuman pluripotent stems cells (hPSCs) in a medium comprising a Gsk3inhibitor, preferably CHIR98014, for at least 24 hours, more preferablyfor 2 days or 3 days, followed by culturing the hPSCs in a mediumcomprising a Porcn inhibitor, preferably Wnt-059 (and lacking the Gsk3inhibitor), for at least 48 hours such that HVPs are generated.Experiments showed that after 24-hour treatment with CHIR-98014, morethan 99% of hPSCs expressed the mesoderm marker Brachyury, and threedays later after treatment with CHIR-98014, more than 95% ofdifferentiatated cells expressed Mesp1, which marks the cardiacmesoderm. Furthermore, 48-hour treatment with Wnt-C59 enhancedventricular progenitor differentiation from mesoderm cells.

Accordingly, with regard to timing of the use of the Gsk3 and Porcninhibitors, typically, at day 0 of culture, the hPSCs are cultured withthe Gsk3 inhibitor, at day 3 of culture the medium is changed to removethe Gsk3 inhibitor and the cells are then cultured with media containingthe Porcn inhibitor through day 5 of culture. HVP generation is optimalbetween days 5 and 7 (inclusive) in culture and peaks at day 6 ofculture. Other non-limiting, exemplary details on culture conditions andtiming of the use of the Gsk3 and Porcn inhibitors are described indetail in Examples 1 and 10.

In Vivo Tissue Engineering

In vivo transplantation studies described in Example 6 and 7 in whichthe human ventricular progenitors (HVPs) were transplanted under thekidney capsule in nude mice document the ability of the HVPs tospontaneously assemble into a large wall of mature, functional, humanventricular muscle on the surface of the kidney capsule. Vascularizationoccurs via a paracrine pathway by calling the murine vasculature to theventricular muscle wall, while a matrix is generated via a cellautonomous pathway from the progenitors themselves. In vivointra-myocardial transplantation studies described in Example 8 in whichthe HVPs were transplanted into the normal murine heart document thatthe HVPs spontaneously migrate to the epicardial surface, where theyexpand, subsequently differentiate, and mature into a wall of humanventricular muscle on the surface of the epicardium. Taken together,these studies show that human ventriculogenesis can occur via acompletely cell autonomous pathway in vivo via purified HVPs, therebyallowing their use in organ-on-organ in vivo tissue engineering.

The human ventricular myocardium has a limited capacity forregeneration, most of which is lost after 10 years of age (Bergmann, O.et al. (2015) Cell 161:1566-1575). As such, new strategies to generateheart muscle repair, regeneration, and tissue engineering approachesduring cardiac injury have been a subject of intense investigation inregenerative biology and medicine (Sahara, M. et al. (2015) EMBO J.34:710-738; Segers, V. F. M. and Lee, R. T. (2008) Nature 451:937-942).Given the need to achieve coordinated vascularization and matrixformation during tissue engineering of any solid organ, the assumptionhas been that the formation of an intact 3-D solid organ in vivo willultimately require the addition of vascular cells and/or conduits, aswell as biomaterials and/or decellularized matrix that will allowalignment and the generation of contractile force (Forbes, S. J. andRosenthal, N. (2014) Nature Med. 20:857-869; Harrison, R. H. et al.(2014) Tissue Eng. Part B Rev. 20:1-16). The complexity of adding thesevarious components to achieve the formation of a functional solid organhas confounded attempts to reduce this to clinical practice (Webber, M.J. et al. (2014) Ann. Biomed. Eng. 43:641-656). Although hPSCs holdgreat promise, to date, it has not been possible to build a pure,vascularized, fully functional, and mature 3-D human ventricular muscleorgan in vivo on the surface of a heart in any mammalian system(Vunjak-Novakovic, G. et al. (2011) Annu. Rev. Biomed. Eng. 13:245-267).

The ability of generate billions of purified HVPs from a renewablesource of either human ES or iPS cell lines represent a new approach tothe generation of functional ventricular muscle in the setting ofadvanced heart failure. The progenitors can be delivered byintramyocardial injection and then self-migrate to the epicardialsurface where they expand and differentiate, losing progenitor markers.Over the course of several week, the cells exit the cell cycle, andproceed to form adult rod-shaped cells that display several independentmarkers of mature ventricular myocardium including the formation of Ttubules, catecholamine responsiveness, loss of automaticity, adult rodshaped conformation with aligned sarcomenric structures, and the abilityto generate force that is comparable to other heart muscle patchesderived from hPSCs differentiated cardiomyocytes (Tulloch, N. L. et al.(2011) Circ. Res. 109:47-59). The scalability of this cell autonomouspathway has allowed the ectopic generation of human ventricular musclethat has a combined thickness in excess of 1.5 cm in thickness,approaching levels that correspond to the human ventricular free wall(Basavarajaiah, S. et al. (2007) Br. J. Sports Med. 41:784-788).

The ability to migrate to the epicardial niche, the site of most of theadult heart progenitors at later stages, is a unique feature of HVPs,and mimics the normal niche of these cells during expansion of theventricular compact zone during ventriculogenesis. Previous studies haveshown that the generation of acute ischemic injury and a breakdown invascular permeability are a pre-requisite for the grafting of relativelysmall numbers of ES cell derived cardiomyocytes into injured myocardium(van Laake, L. W. et al. (2007) Stem Cell Res. 1:9-24; Laflamme, M. A.et al. (2007) Nat. Biotechnol. 25:1015-1024), and even then the survivalrate is low (<5%) (Laflamme, M. A. and Murry, C. E. (2011) Nature473:326-335; Laflamme, M. A. et al. (2005) Am. J. Pathol. 167:663-671).The ability of intra-myocardial HVPs to form an extensive ventricularpatch on the epicardial surface in the absence of acute ischemic injuryprovides a new therapeutic strategy for dilated cardiomyopathy withoutthe need for additional biomaterials, cells, or transfer of exogenousgenes and/or RNAs.

The ability to form a 3-D ventricular muscle wall on the epicardialsurface of the in vivo normal heart is a unique feature of theISL1/FZD4/JAG1 ventricular progenitors as later stage progenitors do notdisplay the ability for the formation of three-dimensional ventriculartissue in either the cardiac or non-cardiac context, emphasizing theimportance of generating a committed ventricular lineage as well aspurifying the specific ventricular progenitor at a specific stage ofventriculogenesis.

Accordingly, the invention provides methods for generating humanventricular tissue in vivo using the HVPs described herein. In oneembodiment, the method comprises transplanting the Jagged 1+ and/orFrizzled 4+ progenitors into an organ of a non-human animal and allowingthe progenitors to grow in vivo such that human ventricular tissue isgenerated. Preferably, the non-human animal is immunodeficient such thatit cannot mount an immune response against the human progenitor cells.In one embodiment, the non-human animal is a mouse, such as animmunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mouse or animmunodeficient SCID-beige mouse (commercially available from CharlesRiver France). In one embodiment, the organ is a kidney (e.g., the cellsare transplanted under the kidney capsule). In another embodiment, theorgan is a heart. In various embodiments, at least 1×10⁶ cells, at least2×10⁶ cells, at least 3×10⁶ cells, at least 4×10⁶ cells, at least 5×10⁶cells, at least 1×10⁷ cells, at least 5×10⁷ cells, at least 1×10⁸ cells,at least 1×10⁹ cells are transplanted.

To obtain HVPs for transplantation, human pluripotent stem cells (hPSCs)can be cultured in vitro under conditions leading to the generation ofHVPs, as described herein (referred to herein as the HVPG protocol).Regarding the timing of transplanting HVPs post in-vitro culture, foroptimal ventricular tissue generation the cells should be transplantedat a stage that can be defined based on the cellular markers expressedby the HVPs at the time of transplantation, determined at days post thestart of culture, which is defined as day 0 of the HVPG protocol. In oneembodiment, the cells are transplanted after the peak of cardiacmesoderm formation, which can be defined as peak expression of themesodermal marker MESP1. Typically, MESP1 expression is between day 2and day 4 of culture (inclusive) and peaks at day 3. In one embodiment,the cells are transplanted at the time corresponding to peak Islet-1expression. Typically, Islet 1 is expressed between day 4 to day 8 ofculture (inclusive) and peaks at day 6 of culture. In one embodiment,the cells are transplanted before the peak of NKX2.5 expression.Typically, NKX2.5 expression starts at day 6 of culture, peaks at day 10of culture and is then maintained afterwards. In one embodiment, thecells are transplanted prior to the peak expression of the downstreamgenes MEF-2 and TBX-1. Typically, these downstream genes are expressedbetween day 5 and day 15 of culture (inclusive) and peaks at day 8 ofculture. In one embodiment, the cells are transplanted prior to theexpression of differentiated contractile protein genes. Typically, theexpression of contractile protein genes (including TNNT2 and MYH6)starts from day 10 of culture onward. In certain embodiments, the cellsare transplanted at a time when two, three or four of the aforementionedmarker patterns are present. In another embodiment, the cells aretransplanted at a time when all five of the aforementioned markerpatterns are present. In one embodiment, the cells are transplantedbetween day 4 to day 8 (inclusive) of culture. In a more preferredembodiment, the cells are transplanted between day 5 to day 7(inclusive) of culture. In the most preferred embodiment, the cells aretransplanted on day 6 of culture.

The transplanted cells can be allowed to grow in the non-human animalfor a suitable period time to allow for the generation of the desiredsize, amount or thickness of ventricular tissue. In various embodiments,the cells are allowed to grow for one week, two weeks. one month, twomonths, three months, four months, five months or six months. The methodcan further comprise harvesting ventricular tissue from the non-humananimal after growth of the cells and differentiation into ventriculartissue.

Methods of Enhancing Cardiac Function

The cardiac ventricular progenitor cells of the invention can be used invivo to enhance cardiac function by transplanting the cells directlyinto the heart. It has now been shown that the Jagged 1+ and/or Frizzled4+ progenitors have the capacity to differentiate into all three typesof cardiac lineage cells (cardiac myocytes, endothelial cells and smoothmuscle cells) (see Example 3). Furthermore, when cultured underconditions that bias toward the ventricular lineage, the Jagged 1+and/or Frizzled 4+ progenitors have now been shown to adopt apredominantly ventricular muscle phenotype when transplanted into thenatural ventricle environment in vivo, demonstrating that theseprogenitor cells “recognize” the ventricular environment and respond anddifferentiate appropriately in vivo. Since damage to the ventricularenvironment is largely responsible for the impaired cardiac function incardiac diseases and disorders, the ability to restore ventricularmuscle cells using the ventricular progenitor cells of the inventionrepresents a significant advance in the art.

Accordingly, in another aspect, the invention provides a method ofenhancing cardiac function in a subject, the method comprisingadministering a pharmaceutical composition comprising the clonalpopulation of Jagged 1+ and/or Frizzled 4+ cardiac ventricularprogenitor cells of the invention to the subject. Preferably, the clonalpopulation is administered directly into the heart of the subject. Morepreferably, the clonal population is administered directly into aventricular region of the heart of the subject. In one embodiment, thepharmaceutical composition administered to the subject comprises theclonal population formulated onto a three dimensional matrix.

The methods of the invention for enhancing cardiac function in a subjectcan be used in a variety of clinical situations involving damage to theheart or reduced or impaired cardiac function. Non-limiting examples ofsuch clinical situations include a subject who has suffered a myocardialinfarction and a subject who has a congenital heart disorder.

Thus, in another aspect, the invention provides a method of treating acardiovascular condition, disease or disorder in a subject, the methodcomprising administering a pharmaceutical composition comprising theclonal population of Jagged 1+ and/or Frizzled 4+ cardiac ventricularprogenitor cells of the invention to the subject. A therapeuticallyeffective amount of cardiac ventricular progenitor cells can beadministered for the treatment of a cardiovascular condition, disease ordisorder. Examples of preferred cardiovascular conditions, diseases ordisorders include coronary artery disease and acute coronary syndrome.

Methods of Use of Cardiac Ventricular Progenitor Cells In Vitro

The cardiac ventricular progenitor cells of the invention can be used invitro in the study of various aspects of cardiac maturation anddifferentiation, in particular in identifying the cells signalingpathways and biological mediators involved in the process of cardiacmaturation and differentiation.

Furthermore, since the Jagged 1+ and/or Frizzled 4+ cardiac ventricularprogenitor cells of the invention are committed to the cardiac lineageand, moreover, are biased toward ventricular differentiation, theseprogenitor cells also are useful for evaluating the cardiac toxicity oftest compounds. All potential new drugs and therapeutics must beevaluated for their toxicity to cardiac cells, before they can be deemedsafe for use in humans. Thus, the ability to assess cardiac toxicity inan in vitro culture system is very advantageous. Accordingly, in anotheraspect, the invention provides a method of screening for cardiactoxicity of test compound, the method comprising

-   -   providing Jagged1+ and/or Frizzled 4+ human cardiac ventricular        progenitor cells;    -   contacting the cells with the test compound; and    -   measuring toxicity of the test compound for the cells,    -   wherein toxicity of the test compound for the cells indicates        cardiac toxicity of the test compound.

In a preferred embodiment, the Jagged 1+ and/or Frizzled 4+ humancardiac ventricular progenitor cells are provided by isolating the cellsaccording to the methods described herein. In a particularly preferredembodiment, the cells are isolated by separating Jagged 1+ and/orFrizzled 4+ cells from a cell culture comprising cardiac progenitorcells using an anti-Jagged 1 and/or anti-Frizzled 4 antibody.Preferably, the cells are isolated using FACS or MACS as describedherein. In yet another embodiment, the Jagged1+ and/or Frizzled 4+ humancardiac ventricular progenitor cells are further cultured anddifferentiation into MLC2v+ ventricular cells prior to contacting withthe test compound.

The toxicity of the test compound for the cells can be measured by oneor more of a variety of different methods for assessing cell viabilityor other physiological functions. Preferably, the effect of the testcompound on cell viability is measured using a standard cell viabilityassay, wherein reduced cell viability in the presence of the testcompound is indicative of cardiac toxicity of the test compound.Additionally or alternatively, cell growth can be measured. Additionallyor alternatively, other indicators of physiological functions can bemeasured, such as cell adhesion, cell signaling, surface markerexpression, gene expression and the like. Similarly, a negative effectof the test compound on any of these indicators of physiologicalfunction is indicative of cardiac toxicity of the test compound.

The invention further provides a method of identifying a compound thatmodulates human cardiac ventricular progenitor cell differentiation, themethod comprising

-   -   providing Jagged1+ and/or Frizzled 4+ human cardiac ventricular        progenitor cells;    -   culturing the cells in the presence or absence of a test        compound;    -   measuring differentiation of the cells in the presence or        absence of the test compound; and    -   selecting a test compound that modulates human cardiac        ventricular progenitor cell differentiation, as compared to        differentiation in the absence of the test compound, to thereby        identify a compound that modulates human cardiac ventricular        progenitor cell differentiation.

In one embodiment, the test compound stimulates human cardiacventricular progenitor cell differentiation. In another embodiment, thetest compound inhibits human cardiac ventricular progenitor celldifferentiation. Differentiation of the cells can be measured by, forexample, measurement of the expression of differentiation markersappearing on the cultured cells over time, as described herein. In apreferred embodiment, the Jagged 1+ and/or Frizzled 4+ human cardiacventricular progenitor cells are provided by isolating the cellsaccording to the methods described herein. In a particularly preferredembodiment, the cells are isolated by separating Jagged 1+ and/orFrizzled 4+ cells from a cell culture comprising cardiac progenitorcells using an anti-Jagged 1 and/or anti-Frizzled 4 antibody.Preferably, the cells are isolated using FACS or MACS as describedherein.

The invention further provides a method of identifying a compound thatmodulates human ventricular cardiomyocyte function, the methodcomprising

-   -   providing Jagged1+ and/or Frizzled 4+ human cardiac ventricular        progenitor cells;    -   culturing the cells in the presence or absence of a test        compound under conditions that    -   generate human ventricular cardiomyocytes;    -   measuring function of the human ventricular cardiomyocytes in        the presence or absence of the test compound; and    -   selecting a test compound that modulates human ventricular        cardiomyocyte function, as compared to function in the absence        of the test compound, to thereby identify a compound that        modulates human ventricular cardiomyoctye function.

In one embodiment, the test compound stimulates human ventricularcardiomyocyte function. In another embodiment, the test compoundinhibits human ventricular cardiomyocyte function. Function of the cellscan be measured by measurement of any suitable indicator of ventricularcell function, including but not limited to, for example, formation of Ttubules, acquisition of adult-rod shaped ventricular cardiomyocytes, andability to generate force in response to electrical stimulation.Suitable assays for measuring such indicators of ventricular cellfunction are known in the art. In a preferred embodiment, the Jagged 1+and/or Frizzled 4+ human cardiac ventricular progenitor cells areprovided by isolating the cells according to the methods describedherein. In a particularly preferred embodiment, the cells are isolatedby separating Jagged 1+ and/or Frizzled 4+ cells from a cell culturecomprising cardiac progenitor cells using an anti-Jagged 1 and/oranti-Frizzled 4 antibody. Preferably, the cells are isolated using FACSor MACS as described herein.

In Vivo Animal Models Using Human Ventricular Progenitor Cells

The development of human iPS and ES cell based models of cardiac diseasehas opened new horizons in cardiovascular drug development anddiscovery. However, to date, these systems have had the limitations ofbeing based on 2D structures in cultured cell systems. In addition, thefetal and immature properties of the cells limit their utility andfidelity to the adult heart. Human cardiac disease, in particular heartfailure, is a complex, multifactorial, multi-organ disease, that isinfluenced by environmental, hormonal, and other key organs that areknown sites for therapeutic targets, such as the kidney. The ability tobuild a mature functional human ventricular organ either ectopically oron the surface of the intact normal murine heart opens up a new in vivomodel system to allow studies that normally could only be assayed on amature human ventricular muscle chamber, such as ventriculararrhythmias, generation of contractile force, fibrosis, and thepotential for regeneration. Accordingly, the option to study humancardiac disease outside of the in vitro tissue culture systems, anddirectly in the context of heart failure in vivo, is now clearlypossible.

Thus, the human ventricular progenitor cells also can be used to createanimal models that allow for in vivo assessment of human cardiac tissuefunction and for in vivo screening of compounds, such as to determinethe cardiac toxicity of a test compound in vivo or to identify compoundsthat modulate human cardiac tissue differentiation or function in vivo.Accordingly, the invention provides methods for testing the effects oftest compounds on human ventricular tissue in vivo using the HVPsdescribed herein. In one embodiment, the method comprises: transplantingJagged 1+ and/or Frizzled 4+ human ventricular progenitors into an organof a non-human animal;

-   -   allowing the progenitors to grow in vivo such that human        ventricular tissue is generated;    -   administering a test compound to the non-human animal; and    -   evaluating the effect of the test compound on the human        ventricular tissue in the non-human animal.

In another embodiment, the method comprises:

-   -   administering a test compound to a non-human animal, wherein the        non-human animal comprises Jagged 1+ and/or Frizzled 4+ human        ventricular progenitors transplanted into an organ of the        non-human animal; and    -   evaluating the effect of the test compound on the Jagged 1+        and/or Frizzled 4+ human ventricular progenitors in the        non-human animal.

In one embodiment, the cardiac toxicity of the test compound isevaluated, for example by measuring the effect of the test compound onthe viability of the human ventricular tissue or the Jagged 1+ and/orFrizzled 4+ human ventricular progenitors in the non-human animal (ascompared to the viability of the tissue or progenitors in the absence ofthe test compound). Cell viability can be assessed by standard methodsknown in the art.

In another embodiment, the ability of a test compound to modulatecardiac differentiation can be evaluated, for example by measuring theeffect of the test compound on the differentiation of the humanventricular tissue or Jagged 1+ and/or Frizzled 4+ progenitors in thenon-human animal (as compared to the differentiation of the tissue orprogenitors in the absence of the test compound). Differentiation of thecells can be measured by, for example, measurement of the expression ofdifferentiation markers appearing on the cells over time.

In another embodiment, the ability of a test compound to modulatecardiac function can be evaluated, for example by measuring the effectof the test compound on the function of the human ventricular tissue orJagged 1+ and/or Frizzled 4+ human progenitors in the non-human animal(as compared to the function of the tissue or progenitors in the absenceof the test compound). Function of the tissue or progenitors can bemeasured by measurement of any suitable indicator of ventricular cellfunction, including but not limited to, for example, formation of Ttubules, acquisition of adult-rod shaped ventricular cardiomyocytes, andability to generate force in response to electrical stimulation.Suitable assays for measuring such indicators of ventricular cellfunction are known in the art.

Preferably, the non-human animal is immunodeficient such that it cannotmount an immune response against the human progenitor cells. In oneembodiment, the non-human animal is a mouse, such as an immunodeficientNOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mouse or an immunodeficient SCID-beigemouse (commercially available from Charles River France). In oneembodiment, the organ is a kidney (e.g., the cells are transplantedunder the kidney capsule). In another embodiment, the organ is a heart.In various embodiments, at least 1×10⁶ cells, at least 2×10⁶ cells, atleast 3×10⁶ cells, at least 4×10⁶ cells, at least 5×10⁶ cells, at least1×10⁷ cells, at least 5×10⁷ cells, at least 1×10⁸ cells, at least 1×10⁹cells are transplanted.

To create the animal models, HVPs for transplantation can be obtained asdescribed above by culturing of hPSCs in vitro under conditions leadingto the generation of HVPs. Regarding the timing of transplanting HVPspost in-vitro culture, for optimal ventricular tissue generation thecells should be transplanted at a stage that can be defined based on thecellular markers expressed by the HVPs at the time of transplantation,determined at days post the start of culture, which is defined as day 0of the HVPG protocol. In one embodiment, the cells are transplantedafter the peak of cardiac mesoderm formation, which can be defined aspeak expression of the mesodermal marker MESP1. Typically, MESP1expression is between day 2 and day 4 of culture (inclusive) and peaksat day 3. In one embodiment, the cells are transplanted at the timecorresponding to peak Islet-1 expression. Typically, Islet 1 isexpressed between day 4 to day 8 of culture (inclusive) and peaks at day6 of culture. In one embodiment, the cells are transplanted before thepeak of NKX2.5 expression. Typically, NKX2.5 expression starts at day 6of culture, peaks at day 10 of culture and is then maintainedafterwards. In one embodiment, the cells are transplanted prior to thepeak expression of the downstream genes MEF-2 and TBX-1. Typically,these downstream genes are expressed between day 5 and day 15 of culture(inclusive) and peaks at day 8 of culture. In one embodiment, the cellsare transplanted prior to the expression of differentiated contractileprotein genes. Typically, the expression of contractile protein genes(including TNNT2 and MYH6) starts from day 10 of culture onward. Incertain embodiments, the cells are transplanted at a time when two,three or four of the aforementioned marker patterns are present. Inanother embodiment, the cells are transplanted at a time when all fiveof the aforementioned marker patterns are present. In one embodiment,the cells are transplanted between day 4 to day 8 (inclusive) ofculture. In a more preferred embodiment, the cells are transplantedbetween day 5 to day 7 (inclusive) of culture. In the most preferredembodiment, the cells are transplanted on day 6 of culture.

The transplanted cells can be allowed to grow in the non-human animalfor a suitable period time to allow for the generation of the desiredsize, amount or thickness of ventricular tissue, prior to administrationof the test compound(s). In various embodiments, the cells are allowedto grow for one week, two weeks. one month, two months, three months,four months, five months or six months.

The present invention is further illustrated by the following examples,which should not be construed as further limiting. The contents offigures and all references, patents and published patent applicationscited throughout this application are expressly incorporated herein byreference.

EXAMPLES Example 1 Generation of Human Isl1+ Cardiomyogenic ProgenitorCells by Modulation of Wnt Signaling in Human Pluripotent Stem Cells

Temporal modulation of canonical Wnt signaling has been shown to besufficient to generate functional cardiomyocytes at high yield andpurity from numerous hPSC lines (Lian, X. et al. (2012) Proc. Natl.Acad. Sci. USA109:E1848-1857; Lian, X. et al. (2013) Nat.Protoc.8:162-175). In this approach, Wnt/β-catenin signaling first isactivated in the hPSCs, followed by an incubation period, followed byinhibition of Wnt/β-catenin signaling. In the originally publishedprotocol, Wnt/β-catenin signaling activation was achieved by incubationwith the Gsk3 inhibitor CHIR99021 (GSK-3 α, IC₅₀=10 nM; GSK-3, βIC₅₀=6.7nM) and Wnt/β-catenin signaling inhibition was achieved by incubationwith the Porcn inhibitor IWP92 (IC₅₀=27 nM). Because we used Gsk3inhibitor and Wnt production inhibitor for cardiac differentiation, thisprotocol was termed GiWi protocol. To improve the efficiency of theoriginal protocol and reduce the potential side effects of the smallmolecules used in the original protocol, a second generation protocolwas developed that uses another set of small molecules with higherinhibition potency. In this second generation GiWi protocol,Wnt/β-catenin signaling activation was achieved by incubation with theGsk3 inhibitor CHIR98014 (CAS 556813-39-9; commercially available from,e.g., Selleckchem) (GSK-3 α, IC₅₀=0.65 nM; GSK-3, βIC₅₀=0.58 nM) andWnt/β-catenin signaling inhibition was achieved by incubation with thePorcn inhibitor Wnt-C59 (CAS 1243243-89-1; commercially available from,e.g., Selleckchem or Tocris) (IC₅₀=74 pM). The Gsk3 inhibitor CHIR98014was used to promote cardiac mesodermal differentiation, whereas thePorcn inhibitor Wnt-C59 was used to enhance ventricular progenitordifferentiation from mesoderm cells.

For cardiomyocyte differentiation via the use of these small molecules,hPSCs were maintained on Matrigel (BD Biosciences) coated plates(Corning) in E8 medium (described in Chen, G. et al. (2011) NatureMethods, 8:424-429; commercially available; STEMCELL Technologies) ormTeSR1 medium (commercially available; STEMCELL Technologies). SuitablehPSCs include induced pluripotent stem cells (iPSCs) such as 19-11-1,19-9-7 or 6-9-9 cells (Yu, J. et al. (2009) Science, 324:797-801) andhuman embryonic stem cells (hESCs), such as ES03 (WiCell ResearchInstitute) and H9 cells (Thomson, J. A. et al. (1998) Science,282:1145-1147).

hPSCs maintained on a Matrigel-coated surface in mTeSR1 medium weredissociated into single cells with Accutase (Life Technologies) at 37°C. for 5 minutes and then seeded onto a Matrigel-coated cell culturedish at 100,000-200,000 cells/cm² in mTeSR1 medium supplemented with 5μM ROCK inhibitor Y-27632 (Selleckchem)(day-2) for 24 hours. Cells werethen cultured in mTeSR1, changed daily. At day 0, cells were thentreated with 1 μM Gsk3 inhibitor CHIR98014 (Selleckchem) for 24 hours(day 0 to day 1) in RPMI/B27-ins (500 ml RPMI with 10 ml B27 supplementwithout insulin). The medium was then changed to the correspondingmedium containing 2 μM the Porcn inhibitor Wnt-C59 (Selleckchem) at day3, which was then removed during the medium change on day 5. Cells weremaintained in RPMI/B27 (stock solution: 500 ml RMPI medium+10 ml B27supplement) starting from day 7, with the medium changed every threedays. This exemplary culturing protocol for generating cardiomyogenicprogenitor cells is illustrated schematically in FIG. 1.

Flow cytometry and immunostaining were preformed to examine theexpression of particular lineage markers. After 24 hour treatment withCHIR-98014, more than 99% of the hPSCs expressed the mesoderm markerBrachyury. Three days after treatment with CHIR-98014, more than 95% ofdifferentiated cells expressed Mespl, which marks the cardiac mesoderm.The culture protocol not only allowed the cells to synchronouslydifferentiate into the cardiac mesodermal lineage, but also reproduciblygenerated more than 90% of ventricular myocytes after 14 days ofdifferentitation, as determined by cTnT flow cytometry andelectrophysiology analysis.

To further assess cardiac differentiation of the hPSCs over time,Western blot analysis was performed on days 0-7 and d11 to examine theexpression of Isl1 and Nkx2.5 (cardiomyogenic progenitor markers) andcTnI (a cardiac myocyte marker). Cells were lysed in M-PER MammalianProtein Extraction Reagent (Pierce) in the presence of Halt Protease andPhosphatase Inhibitor Cocktail (Pierce). Proteins were separated by 10%Tris-Glycine SDS/PAGE (Invitrogen) under denaturing conditions andtransferred to a nitrocellulose membrane. After blocking with 5% driedmilk in TBST, the membrane was incubated with primary antibody overnightat 4° C. The membrane was then washed, incubated with ananti-mouse/rabbit peroxidase-conjugated secondary antibody at roomtemperature for 1 hour, and developed by SuperSignal chemiluminescence(Pierce). The results are shown in FIG. 2. During cardiacdifferentiation of hPSCs, Isl1 expression started on day 4 and increasedto its maximum expression on day 6, whereas NKx2.5 only started toexpress on day 6 and reached its maximum expression after day 10.Cardiomyoctes (cTn1+ cells) were not induced until day 11 ofdifferentiation.

In addition, immunostaining of the day 6 cells was performed for Isl1expression. Cells were fixed with 4% formaldehyde for 15 minutes at roomtemperature and then stained with primary (anti-Isl1) and secondaryantibodies in PBS plus 0.4% Triton X-100 and 5% non-fat dry milk(Bio-Rad). Nuclei were stained with Gold Anti-fade Reagent with DAPI(Invitrogen). An epifluorescence microscope (Leica DM IRB) with aQImaging® Retiga 4000R camera was used for imaging analysis. The resultsshowed substantial numbers of Isl1+ cells.

Flow cytometry analysis of day 6 cells for Isl1 expression also wasperformed. Cells were dissociated into single cells with Accutase for 10minutes and then fixed with 1% paraformaldehyde for 20 minutes at roomtemperature and stained with primary and secondary antibodies in PBS0.1% Triton X-100 and 0.5% BSA. Data were collected on a FACSCaliberflow cytometer (Beckton Dickinson) and analyzed using FloJo. Theresults, shown in FIG. 3, showed that more than 95% of cells expressedIsl1 at this stage. In summary, this example provides a protocol forhuman ventricular progenitor generation (HVPG protocol) that allows forthe large-scale production of billions of Isl1+ human HPVs efficientlywithin 6 days.

Example 2 Identification of Jagged 1 as a Cell Surface Marker of CardiacProgenitor Cells

To profile the transcriptional changes that occur during the cardiacdifferentiation process at a genome-scale level, RNA sequencing(RNA-seq) was performed at different time points followingdifferentiation to build cardiac development transcriptional landscapes.We performed RNA-seq experiments on day 0 to day 7 samples, as well asday 19 and day 35 samples (two independent biological replicates pertime point). Two batches of RNA-seq (100 by and 50 by read length) wereperformed using the illumine Hiseq 2000 platform. In total, 20 sampleswere examined. Bowtie and Tophat were used to map our reads into areference human genome (hg19) and we calculate each gene expression(annotation of the genes according to Refseq) using RPKM method (Readsper kilobase transcript per million reads). Differentiation of hPSCs tocardiomyocytes involves five major cell types: pluripotent stem cells(day 0), mesoderm progenitors (day 1 to day 2), cardiac mesoderm cells(day 3 to day 4), heart field progenitors (day 5, day 6 and day 7), andcardiomyocytes (day 10 after).

Molecular mRNA analysis of cardiac differentiation from hPSCs using theHVPG protocol revealed dynamic changes in gene expression, withdown-regulation of the pluripotency markers OCT4, NANOG and SOX2 duringdifferentiation. Induction of the primitive streak-like genes T andMIXL1 occurred within the first 24 hours following CHIR-98014 addition,and was followed by upregulation of the cardiac mesodermal marker MESP1on day 2 and day 3. Expression of the cardiac muscle markers TNNT2,TNNC1, MYL2, MYL7, MYH6, MYH7 and IRX4 was detected at later stage ofdifferentiation (after day 10).

By this analysis, genes enriched at each differentiation stage,including mesoderm cells, cardiac progenitors and cardiomyocytes, wereidentified. Mesoderm cells, which are related to day 1 differentiatedcells, express brachyury. We identified potential surface markers formesoderm cells, including: FZD10, CD48, CD1D, CD8B, IL15RA, TNFRSF1B,TNFSF13, ICOSLG, SEMA7A, SLC3A2, SDC1, HLA-A. Through similar analysis,we also identified surface markers for cardiac mesoderm mesp1 positivecells, including: CXCR4, ANPEP, ITGAS, TNFRSF9, FZD2, CD1D, CD177,ACVRL1, ICAM1, L1CAM, NGFR, ABCG2, FZD7, TNFRSF13C, TNFRSF1B.

Consistent with western blot analysis, ISL1 mRNA was expressed as earlyas day 4 and peaked on day 5, one day before its protein expressionreached its peak. On day 5 of differentiation (the cardiac progenitorstage, isl1 mRNA expression maximum on day 5, isl1 protein expressionmaximum on day 6), the day 5 enriched genes were compared with ananti-CD antibody array (a panel of 350 known CD antibodies) and a numberof potential cell-surface protein markers were identified. We identifiedmany cell-surface proteins expressed at this stage, including: FZD4,JAG1, PDGFRA, LIFR (CD118), TNFSF9, FGFR3.

The cell surface protein Jagged 1 (JAG1) and Frizzled 4 (FZD4) wereselected for further analysis. Jagged 1 expression was further studiedas described below and in Examples 3 and 4. Frizzled 4 expression wasfurther studied as described in Example 5.

Firstly, the expression of Is11 and Jag1 was profiled using the doublestaining flow cytometry technique. Flow cytometric analysis was carriedout essentially as described in Example 1, using anti-Isl1 and anti-Jag1antibodies for double staining. The results are shown in FIG. 4. Jagged1 expression was found to trace the expression of Islet 1 and on day 6of differentiation, all of the Islet 1 positive cells also expressedJagged 1, and vice versa. Because of the co-expression pattern of thesetwo markers, a Jagged 1 antibody was used to enrich the 94.1% Islet1+cells differentiated population to 99.8% purity of Islet1+ Jagged1+cells.

It also was confirmed that Islet 1 is an earlier developmental gene thanthe Nkx2.5 gene using double immunostaining of ISL1 and NKX2.5expression in HVPs. The purified HVPs uniformly express the ISL1 gene,but at this stage, only a few of the cells started to express Nkx2.5.

Furthermore, immunostaining with both anti-Isl1 and anti-Jag 1 wasperformed, essentially as described in Example 1, on week 4 human fetalheart tissue, neonatal heart tissue and 8-year old heart tissue. Theresults revealed that in the in vivo fetal heart, all of the Islet 1positive cells also expressed Jagged 1. However, the neonatal heart and8-year old heart did not express Islet 1 or Jagged 1. In the ventricleof week 4 human fetal heart, cardiac Troponin T (cTnT) staining revealedvisible sarcomere structures. In addition, over 50% of ventricular cellsin the week 4 fetal heart expressed both Islet1 and Jagged1, which wasmarkedly decreased during subsequent maturation, with the loss ofexpression of both Islet1 and Jagged1 in the ventricular muscle cells ofthe human neonatal hearts.

The above-described experiments demonstrate that Jagged 1 is a cellsurface marker for Islet 1 positive cardiomyogenic progenitor cells.

Example 3 Clonal Differentiation of Isl1+ Jag1+ Cardiac Progenitor Cells

To characterize the clonal differentiation potential of Isl1+ Jag1+cells, cardiomyogenic progenitor cells were generated by the culturingprotocol described in Example 1, and one single Isl1+ Jag1+ cell wasseeded into one well of a Matrigel-coated 48-well plate. Cells werepurified with antibody of Jag 1 and then one single cell was seeded intoone well. The single cells were then cultured for 3 weeks in CardiacProgenitor Culture (CPC) medium (advanced DMEM/F12 supplemented with 2.5mM GlutaMAX, 100 μg/ml Vitamin C, 20% Knockout Serum Replacement).

Immunostaining of the 3-week differentiation cell population was thenperformed with three antibodies: cardiac troponin I (cTn1) forcardiomyocytes, CD144 (VE-cadherin) for endothelial cells and smoothmuscle actin (SMA) for smooth muscle cells. The results showed that thesingle cell-cultured, Isl1+ Jag1+ cells gave rise to cTnI positive andSMA positive cells, but not VE-cadherin positive endothelial cells,indicating these generated Islet1+ cells are heart muscle progenitorsthat have limited differentiation potential to endothelial lineages.Purified Islet1+ Jagged1+ cells differentiated with the HVPG protocolfrom human induced pluripotent stem cells (iPSC 19-9-11 line) alsoshowed similar in vitro differentiation potential and predominantlydifferentiate to cTn1+ SMA+ cells, but not VE-cadherin+ cells. Over thecourse of several weeks, the cells expressed the ventricular specificmarker MLC2v, indicating that the initial ISL1+ subset was alreadycommitted to the ventricular cell fate. Because of the limited vasculardifferentiation potential of Islet1+ cells generated using the HVPGprotocol, these generated Islet1+ cells might represent a distinctprogenitor population from the previously reported KDR+ population(Yang, L. et al. (2008) Nature 453:524-528) or multipotent ISL1+ cells(Bu, L. et al. (2009) Nature 460:113-117; Moretti, A. et al. (2006) Cell127:1151-1165), which can give rise to all three lineages ofcardiovascular cells.

These results demonstrated that the Isl1+ Jag1+ cardiomyogenicprogenitor cells can be successfully cultured in vitro from a singlecell to a significantly expanded cell population (1×10⁹ cells orgreater) that contains all three types of cardiac lineage cells, with apredominance of cardiomyocytes. Furthermore, these cells can be culturedin vitro for extended periods of time, for at least 2-3 weeks, and evenfor months (e.g., six months or more). Since the cardiomyogenicprogenitor cells gradually differentiate into cardiomyocytes, which donot proliferate, a culture period of approximately 2-3 weeks ispreferred.

Example 4 In Vivo Developmental Potential of Isl1+ Jag1+ CardiacProgenitor Cells'

The ES03 human embryonic stem cell (hESC) line (obtained from WiCellResearch Institute) expresses green fluorescent protein (GFP) driven bythe cardiac-specific cTnT promoter. ES03 cells were used to generateIsl1+ Jag1+ cardiomyogenic progenitor cells using the culturing protocoldescribed in Example 1. The Isl1+ Jag1+ cardiomyogenic progenitor cellswere transplanted into the hearts of severe combined immunodeficient(SCID) beige mice to document their developmental potential in vivo.

Briefly, Isl1+ Jag1+ cells were injected (1,000,000 cells per recipient)directly into the left ventricular wall of NOD/SCID-gamma mice in anopen-chest procedure. Hearts were harvested 2-3 weeks post surgery,fixed in 1% PFA and sectioned at 10 μm (n=12).

Histological analyses of the hearts of the transplanted mice revealedthe presence of GFP+donor cells, detected by epifluorescence and bystaining with an anti-GFP antibody, demonstrating that the Isl1+ Jag1+cardiomyogenic progenitor cells were capable of differentiating intocardiomyocytes when transplanted in vivo.

The Isl1+ Jag1+ cardiomyogenic progenitor cells were also transplanteddirectly into infarcted hearts of SCID beige mice (“injured mice”), ascompared to similarly transplanted normal mice. When analyzed two weekslater, injured mice transplanted with the Isl1+ Jag1+ cardiomyogenicprogenitor cells had a larger graft size than the normal mice similarlytransplanted, demonstrating the cardiomyocyte regeneration capacity ofthe Isl1+ Jag1+ cardiomyogenic progenitor cells in vivo.

Example 5 Identification of Frizzled 4 as a Cell Surface Marker ofCardiac Progenitor Cells

As described in Example 2, Frizzled 4 (FZD4) was identified by RNA-seqanalysis as being expressed in cardiac progenitor cells. Thus, toconfirm FZD4 as a cell surface marker of cardiac progenitor cells, FZD4expression was assessed during cardiac differentiation via Western blotanalysis. The results, as shown in FIG. 5, demonstrated that FZD4 wasnot express in pluripotent stem cells and the first 3 daysdifferentiated cells. However, FZD4 started to express on day 4 andmaximize its expression on day 5 of expression.

In order to quantify the co-expression pattern of FZD4 and Is11 at thesingle cell level, FACS analysis was performed. As shown in FIG. 6, onday 5 of differentiation, more than 83% of cells express both isll andFZD4, demonstrating that FZD4 is a cell surface marker for isll positivecells during cardiac progenitor differentiation using the GiWi protocol.

In order to confirm that both JAG1 and FZD4 were indeed co-expressedwith ISL1 on the human ventricular progenitor cells, tripleimmunofluorescence analysis of day 6 differentiated cells from hPSCs wasperformed with antibodies to Islet 1, Jagged 1 and Frizzled 4. Thetriple staining experiment demonstrated that Isl1+cells expressed bothJagged 1 and Frizzled 4.

Example 6 Human Ventricular Progenitors (HPVs) Generate a 3-DVentricular Heart Muscle Organ In Vivo

The building of the ventricular heart muscle chamber is one of the mostcritical and earliest steps during human organogenesis, and requires aseries of coordinated steps, including migration, proliferation,vascularization, assembly, and matrix alignment. To test the capacity ofHVPs to drive ventriculogenesis in vivo, we transplanted purified HVPsor unpurified HVPs (92.0±1.9% ISL1+) under the kidney capsule ofimmunocompromised mice. After 2 months post-transplantation, animalstransplanted with unpurified HVPs formed tumors, resulting in a tumorformation efficiency of 100% (100%, 4/4), whereas animals transplantedwith purified HVPs did not form any tumors (0%, 0/10).

The engrafted kidneys with purified HVPs were further assayed forhistological analysis. Hematoxylin and Eosin (H&E) staining revealed anorgan that exceeded 0.5 cm in length with more than 1 mm thickness onthe surface of the mouse kidney, and that uniformly expressed theventricular specific marker MLC2v (O'Brien, T. X. et al. (1993) Proc.Natl. Acad. Sci. USA 90:5157-5161). The resulting human muscle organ wasfully vascularized and red blood cells could be detected in the bloodvessels. Analysis of cTnT, MLC2v, and MLC2a immunostaining furtherrevealed that the transplanted HVPs not only differentiated into cardiacmuscle cells (cTnT+cells), but also further mature to becomeMLC2v+ventricular myocytes that are negative for MLC2a expression. Theresulting ventricular muscle organ is fully vascularized by murinederived vascular cells, consistent with the notion that itsvascularization occurred via paracrine cues derived from the HVPs.

The blood vessel structured was revealed by immunostaining analysis ofantibodies directed against VE-cadherin and smooth muscle actinexpression. In addition, using a human specific monoclonal lamininantibody targeting laminin y-1 chain, the HVPs secreted their own humanlaminin as their extracellular matrix (the mouse kidney region isnegative for human laminin immunostaining). In addition, we found humanfibronectin expression is restricted to areas near the blood vesselsusing a monoclonal human fibronectin antibody.

To assess the capacity of late stage cardiac cells to driveventriculogenesis, NKX2.5+cells (day 10 after differentiation) weretransplanted under the kidney capsule of immunocompromised NSG mice. Atthree weeks post-transplantation, animals transplanted with NKX2.5+cellsdid not form any visible human muscle graft, indicating that HVPs losetheir ability for in vivo ventriculogenesis following peak Islet-1expression.

Taken together, these studies indicate that the HVPs can synthesize andrelease their own cardiac laminin-derived matrix, as well as fibronectinwhich serves to stabilize the vasculature to the nascent ventricularorgan.

Example 7 HVPs Create a Mature, Functioning Ventricular Muscle Organ InVivo via a Cell Autonomous Pathway

One of the critical limitations for the utility of hPSCs for studies ofhuman cardiac biology and disease is their lack of maturity andpersistence of expression of fetal isoforms. To determine if the HVPderived organs could become functional mature ventricular muscle, longterm transplantation studies were performed followed by detailedanalyses of a panel of well accepted features of adult ventricularmyocardium including formation of T tubules (Brette, F. and Orchard, C.(2003) Circ. Res. 92:1182-1192; Marks, A. R. (2013) J. Clin. Invest.123:46-52), ability to generate force comparable to other studies ofengineered ventricular tissue, loss of automaticity, and acquisition ofadult-rod shaped ventricular cardiomyocytes.

After 5 months post-transplantation of purified HVPs, no tumors formedin all of our animals. Animals were sacrificed and the engrafted kidneyswere removed for further analysis. The 5-month human graft was ahemisphere structure with the radius of 0.4 cm (diameter of 0.8 cm). Thevolume for the 5-month human graft was around 0.13 cm³ for one kidney, avolume that suggests feasibility for generating human ventricular musclethat achieves a thickness comparable to the in vivo human adult heart.Rod-shaped mature human ventricular myocytes were observed in the humanmuscle organ. In addition, muscle trips taken from our mature humanmuscle organ generated forces (0.36±0.04 mN) in response to electricstimulation and increased their force generation after treatment with aβ-adrenergic agonist isoprenaline (0.51±0.02 mN, p<0.05 compared tocontrol). Taken together, these studies indicate that the HVPs arecapable of generating a fully functional, mature human ventricularmuscle organ in vivo via a cell autonomous pathway, i.e., without theaddition of other cells, genes, matrix proteins, or biomaterials.

Example 8 HVPs Migrate Towards an Epicardial Niche and SpontaneouslyForm a Human Ventricular Muscle Patch on the Surface of a Normal MurineHeart In Vivo

The epicardium is a known niche for heart progenitors, driving thegrowth of the ventricular chamber during compact zone expansion, as wellas serving as a home to adult epicardial progenitors that can expandafter myocardial injury and that can drive vasculogenesis in response toknown vascular cell fate switches, such as VEGF (Giordano, F. J. et al.(2001) Proc. Natl. Acad. Sci. USA 98:5780-5785; Masters, M. and Riley,P. R. (2014) Stem Cell Res. 13:683-692; Zangi, L. et al. (2013) Nat.Biotechnol. 31:898-907). To determine if the HVPs might migratespontaneously to the epicardial surface of the normal heart, purifiedgreen fluorescent protein (GFP)-labeled HVPs were injectedintra-myocardially into the hearts of immunocompromised mice. After oneweek or one month post-transplantation, animals were sacrificed and theengrafted hearts were removed for histology. After one weekpost-transplantation, the majority of GFP+ cells were retained in themyocardium. However, almost all the GFP+ cells migrated to theepicardium after one month post-transplantation. In addition, GFP+ cellswere ISL1+ and Ki67+ after one week post-transplantation.

In order to trace the differentiation potential of Islet1+ cells, thepurified ISL1+ JAG1+ cells generated from a cTnT promoter driven greenfluorescent protein (GFP)-expressing hESC line (H9-cTnT-GFP) weretransplanted into the hearts of severe combined immunodeficient (SCID)beige mice to document their developmental potential in vivo. One monthafter transplantation of Isl1+ Jag1+ cells directly into the ventricleof the hearts of SCID beige mice, Hematoxylin and eosin stainingrevealed a human muscle strip graft present in the epicardium of themurine heart. In addition, immunohistological analyses revealed thepresence of GFP+ donor cells detected by epifluorescence and by stainingwith an anti-GFP antibody, More importantly, when analysed withantibodies of MLC2v and MLC2a, the grafted human muscle strip ispositive for MLC2v (100% of cells +), and negative for the atrial markerMLC2a, indicating the transplanted ISL1+ cells not only furtherdifferentiated to cardiac muscle cells, but also became ventricularmuscle cells. Taken together, these studies indicate that the HVPs canmigrate to an epicarial niche, where they expand, and subsequentlydifferentiate in to a homogenous ventricular muscle patch, again withoutthe addition of exogenous cells, genes, matrices, or hiomaterials.

Example 9 Additional Experimental Materials and Methods

In this example, additional details on the experimental materials andmethods used in Examples 1-8 are provided.

Maintenance of hPSCs

hESCs (ES03, H9) and human iPSCs (19-9-11) were maintained on Matrigel(BD

Biosciences) coated plates in mTeSR1 medium (STEMCELL Technologies)according to previous published methods (Lian, X. et al. (2013) Nat.Proc. 8:162-175; Lian, X. et al. (2013) Stem Cells 31:447-457).

Human Ventricular Progenitor Generation (HVPG) protocol

hPSCs maintained on a Matrigel-coated surface in mTeSR1 were dissociatedinto single cells with Accutase at 37° C. for 10 min and then seededonto a Matrigel-coated cell culture dish at 100,000-200,000 cell/cm² inmTeSR1 supplemented with 5 μM ROCK inhibitor Y-27632 (day-2) for 24hours. At day-1, cells were cultured in mTeSR1. At day 0, cells weretreated with 1 μM CHIR-98014 (Selleckchem) in RPMI supplemented with B27minus insulin (RPM1/B27-ins) for 24 hours (day 0 to day 1), which wasthen removed during the medium change on day 1. At day 3, half of themedium was changed to the RPM1/B27-ins medium containing 2 μM Wnt-C59(Selleckchem), which was then removed during the medium change on day 5.At day 6, cells were dissociated into single cells and purified withanti-JAG1 or anti-FZD4 antibody.

RNA-seq Library Construction

RNA was isolated (RNeasy Mini kit, Qiagen), quantified (Qubit RNA AssayKit, Life Technologies) and quality controlled (BioAnalyzer 2100,Agilent). RNA (800 ng) from each sample was used as input for theIllumina TruSeq mRNA Sample Prep Kit v2 (Illumina) and sequencinglibraries were created according to the manufacturer's protocol.Briefly, poly-A containing mRNA molecules were purified using poly-Toligo-attached magnetic beads.

Following purification, the mRNA was fragmented and copied into firststrand complementary DNA using random primers and reverse transcriptase.Second strand cDNA synthesis was then done using DNA polymerase I andRNase H. The cDNA was ligated to adapters and enriched with PCR tocreate the final cDNA library. The library was pooled and sequenced on aHiSeq 2000 (Illumina) instrument per the manufacturer's instructions.

RNA-seq Data Processing

The RNA-seq reads were trimmed and mapped to the hg19 reference usingTophat 2. On average, approximately 23 million reads were generated persample, and 76% of these reads were uniquely mapped. Expression levelsfor each gene were quantified using the python script rpkmforgenes andannotated using RefSeq. Genes without at least one sample with at leastten reads were removed from the analysis. Principle Component Analysisand heatmaps were constructed using the R and Gene-E respectively.

Transplantation

Aliquots of 2 million purified HVPs were collected into an eppendorftube. Cells were spun down, and the supernatant was discarded. Each tubeof cells was transplanted under the kidney capsule, orintra-myocardially injected into the heart of the immunodeficient mice,NOD.Cg-Prkdcscid Il2rgtm1Wj1/SzJ or SCID-Beige respectively (CharlesRiver France), following a previously described protocol (Shultz, L.D.et al. (2005) J. Immunol. 174:6477-6489). Engrafted Kidneys or heartsare harvested at various time intervals for histological andphysiological analysis.

Flow Cytometry

Cells were dissociated into single cells with Accutase for 10 min andthen fixed with 1% paraformaldehyde for 20 min at room temperature andstained with primary and secondary antibodies in PBS plus 0.1% TritonX-100 and 0.5% BSA. Data were collected on a FACSCaliber flow cytometer(Beckton Dickinson) and analyzed using FlowJo.

Immunostaining

Cells were fixed with 4% paraformaldehyde for 15 min at room temperatureand then stained with primary and secondary antibodies in PBS plus 0.4%Triton X-100 and 5% non-fat dry milk (Bio-Rad). Nuclei were stained withGold Anti-fade Reagent with DAPI (Invitrogen). An epifluorescencemicroscope and a confocal microscope (ZEISS, LSM 700) were used forimaging analysis.

Western Blot Analysis

Cells were lysed in M-PER Mammalian Protein Extraction Reagent (Pierce)in the presence of Halt Protease and Phosphatase Inhibitor Cocktail(Pierce). Proteins were separated by 10% Tris-Glycine SDS/PAGE(Invitrogen) under denaturing conditions and transferred to anitrocellulose membrane. After blocking with 5% dried milk in TBST, themembrane was incubated with primary antibody overnight at 4° C. Themembrane was then washed, incubated with an anti-mouse/rabbitperoxidase-conjugated secondary antibody at room temperature for 1 hour,and developed by SuperSignal chemiluminescence (Pierce).

Electrophysiology (Patch Clamping)

Beating ventricular myocyte clusters were microdissected and replatedonto glass coverslips before recording. Action potential activity wasassessed using borosilicate glass pipettes (4-5

M Ohm resistance) filled with intracellular solution consisting of 120mM K D-gluconate, 25 mM KCl, 4 mM MgATP, 2 mM NaGTP, 4 mMNa2-phospho-creatin, 10 mM EGTA, 1 mM CaCl2, and 10 mM HEPES (pH 7.4adjusted with HCl at 25° C.). Cultured cardiomyocytes seeded oncoverslip dishes were submerged in extracellular solution (Tyrode'ssolution) containing 140 mM NaC1, 5.4 mM KCl, 1 mM MgC12, 10 mM glucose,1.8 mM CaC12, and 10 mM HEPES (pH 7.4 adjusted with NaOH at 25° C.).Spontaneous action potentials were recorded at 37° C. using patch clamptechnique (whole-cell, current clamp configuration) performed using aMulticlamp 700B amplifier (Molecular Devices, CA, USA) software low-passfiltered at 1 kHz, digitized and stored using a Digidata 1322A andClampex 9.6 software (Molecular Devices, Calif., USA).

Statistics

Data are presented as mean±standard error of the mean (SEM). Statisticalsignificance was determined by Student's t-test (two-tail) between twogroups. P<0.05 was considered statistically significant.

Example 10 Xeno-Free Human Ventricular Progenitor DifferentiationProtocol

In this example, an alternative differentiation protocol fordifferentiation of human ventricular progenitors is provided, whichutilizes a defined, xeno-free culture medium, Essential 8. The Essential8 medium was developed for growth and expansion of human pluripotentstem cells (hPSCs) and is described further in Chen, G. et al. (2011)Nat. Methods 8:424-429 (referred to therein as “E8” medium).

hPSCs maintained on a Vitronectin (or Laminin 521)-coated surface inEssential 8 medium were dissociated into single cells with Versenesolution at 37° C. for 10 min and then seeded onto a Vitronectin (orLaminin 521)-coated cell culture dish at 100,000-200,000 cell/cm2 inEssential 8 medium supplemented with 5 μM ROCK inhibitor Y-27632 (day-2) for 24 hours. At day -1, cells were cultured in Essential 8 medium.At day 0, cells were treated with 0.5 μM CHIR-98014 in RPMI for 24 hours(day 0 to day 1), which was then removed during the medium change onday 1. At day 3, half of the medium was changed to the RPMI mediumcontaining 0.5 μM Wnt-C59, which was then removed during the mediumchange on day 5. At day 6, cells (human ventricular progenitors) weredissociated into single cells and purified with anti-JAG1 or anti-FZD4antibody.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method for isolating human cardiac ventricular progenitor cells,the method comprising: contacting a culture of human cells containingcardiac progenitor cells with one or more agents reactive with Jagged 1and/or Frizzled 4; and separating Jagged 1 reactive positive cellsand/or Frizzled 4 reactive positive cells from non-reactive cells tothereby isolate human cardiac ventricular progenitor cells. 3.-5.(canceled)
 6. The method of claim 1, wherein the agent reactive withJagged 1 is an anti-Jagged 1 antibody and/or the agent reactive withFrizzled 4 is an anti-Frizzled 4 antibody. 7.-8. (canceled)
 9. Themethod of claim 1, wherein the Jagged 1 reactive positive cells and/orFrizzled 4 reactive positive cells are separated from the non-reactivecells by fluorescence activated cell sorting.
 10. The method of claim 1,wherein the human cardiac ventricular progenitor cells are furthercultured and differentiated such that they are MLC2v positive.
 11. Amethod for isolating human cardiac ventricular progenitor cells, themethod comprising: culturing human pluripotent stem cells underconditions that generate cardiac progenitor cells to obtain a culture ofcells; contacting the culture of cells with one or more agents reactivewith Jagged 1 and/or Frizzled 4; and separating Jagged 1 reactivepositive cells and/or Frizzled 4 reactive positive cells fromnon-reactive cells to thereby isolate human cardiac ventricularprogenitor cells.
 12. The method of claim 11, wherein the culture ofcells is further contacted with an agent reactive with Islet 1; andJagged 1 reactive/Islet 1 reactive positive cells, Frizzled 4reactive/Islet 1 reactive positive cells or Jagged 1 reactive/Frizzled 4reactive/Islet 1 reactive positive cells are separated from non-reactivecells to thereby isolate human cardiac ventricular progenitor cells.13.-15. (canceled)
 16. The method of claim 11, wherein the agentreactive with Jagged 1 is an anti-Jagged 1 antibody and/or the agentreactive with Frizzled 4 is an anti-Frizzled 4 antibody. 17.-18.(canceled)
 19. The method of claim 11, wherein the Jagged 1 reactivepositive cells and/or Frizzled 4 reactive positive cells are separatedfrom the non-reactive cells by fluorescence activated cell sorting. 20.The method of claim 11, wherein the human cardiac ventricular progenitorcells are further cultured and differentiated such that they are MLC2vpositive.
 21. A method for obtaining a clonal population of humancardiac ventricular progenitor cells, the method comprising: isolating asingle Jagged 1+ and/or Frizzled 4+ human cardiac ventricular progenitorcell; and culturing the single Jagged 1+ and/or Frizzled 4+ humancardiac ventricular progenitor cell under conditions such that the cellis expanded to at least 1×10⁹ cells to thereby obtain a clonalpopulation of human cardiac ventricular progenitor cells.
 22. The methodof claim 21, wherein the single Jagged 1+ and/or Frizzled 4+ humancardiac ventricular progenitor cell is Islet 1 positive, Nkx2.5 negativeand flk1 negative at the time of initial culture.
 23. The method ofclaim 21, wherein the single Jagged 1+ and/or Frizzled 4+ human cardiacventricular progenitor cell is isolated by fluorescence activated cellsorting.
 24. The method of claim 21, wherein the single Jagged 1+ and/orFrizzled 4+ human cardiac ventricular progenitor cell is isolated usingone or more reagents reactive with Jagged 1 and/or Frizzled
 4. 25. Themethod of claim 24, wherein the reagent reactive with Jagged 1 orFrizzled 4 is an anti-Jagged 1 antibody or an anti-Frizzled 4 antibody.26. The method of claim 21, wherein the single Jagged 1+ and/or Frizzled4+ human cardiac ventricular progenitor cell is cultured in CardiacProgenitor Culture (CPC) medium.
 27. The method of claim 21, wherein thesingle Jagged 1+ and/or Frizzled 4+ human cardiac ventricular progenitorcell is cultured under conditions such that the cell is biased towardventricular differentiation.
 28. The method of claim 21, wherein thesingle Jagged 1+ and/or Frizzled 4+ human cardiac ventricular progenitorcell is expanded to at least 10×10⁹ cells.
 29. A clonal population of atleast 1×10⁹ Jagged 1+ and/or Frizzled 4+ human cardiac ventricularprogenitor cells obtained by the method of claim
 21. 30. A clonalpopulation of at least 10×10⁹ Jagged 1+ and/or Frizzled 4+ human cardiacventricular progenitor cells obtained by the method of claim
 21. 31. Amethod of enhancing cardiac function in a subject, the method comprisingadministering a pharmaceutical composition comprising the clonalpopulation of claim 29 to the subject.
 32. The method of claim 31,wherein the clonal population is administered directly into the heart ofthe subject.
 33. The method of claim 31, wherein the subject hassuffered a myocardial infarction.
 34. The method of claim 33, whereinthe subject has a congenital heart disorder.
 35. The method of claim 32,wherein the clonal population is administered directly into aventricular region of the heart of the subject.
 36. The method of claim3, wherein the pharmaceutical composition comprises the clonalpopulation formulated onto a two dimensional or three dimensionalmatrix.
 37. A method for generating human ventricular tissue comprisingtransplanting Jagged 1+ and/or Frizzled 4+ human cardiac ventricularprogenitor cells into an organ of a non-human animal; and allowing theprogenitor cells to grow in vivo such that human ventricular tissue isgenerated.
 38. The method of claim 37, wherein the non-human animal isan immunodeficient mouse.
 39. The method of claim 37, wherein the organis a kidney or a heart.
 40. The method of claim 37, wherein the cellsare transplanted at a time when one, two, three, four or five of thefollowing cell marker patterns are present: (i) after peak of cardiacmesoderm formation; (ii) at time of peak Islet-1 expression; (iii)before peak of NKX2.5 expression; (iv) before peak expression ofdownstream genes MEF-2 and TBX-1; and (v) before expression ofdifferentiated contractile protein genes.
 41. The method of claim 37,wherein the cells are transplanted between day 5 and day 7 (inclusive)of in vitro culture of human pluripotent stem cells under conditions togenerate human ventricular progenitor cells.
 42. The method of claim 41,wherein the cells are transplanted on day 6 of in vitro culture of humanpluripotent stem cells under conditions to generate human ventricularprogenitor cells.
 43. A method of screening for cardiac toxicity of testcompound, the method comprising providing Jagged1+ and/or Frizzled 4+human cardiac ventricular progenitor cells; contacting the cells withthe test compound; and measuring toxicity of the test compound for thecells, wherein toxicity of the test compound for the cells indicatescardiac toxicity of the test compound.
 44. A method of generating humanventricular progenitors (HVPs) comprising: culturing human pluripotentstems cells (hPSCs) in a medium comprising CHIR98014 such that cellsexpressing cardiac mesodermal markers are generated, and culturing thecells expressing cardiac mesodermal markers in a medium comprisingWnt-C59 such that HVPs are generated.